(EFT) Treatment

32 Pages·2016·0.75 MB·English

ORIGINAL RESEARCH
Differential Gene Expression after Emotional
Freedom Techniques (EFT) Treatment: A Novel Pilot
Protocol for Salivary mRNA Assessment
Marjorie E. Maharaj, Department of Applied Psychology,
Akamai University, Hilo, HI
Abstract
Biopsychology is a rapidly expanding field evidence-based practice combining acupres-
of study since the completion of the Human sure and cognitive exposure. The control treat-
Genome Project in 2003. There is little data ment was non-therapeutic social interaction.
measuring the effect of psychotherapeutic To establish a baseline, participants received
interventions on gene expression, due to the the control first, followed a week later by EFT.
technical, logistical, and financial require- Analysis of samples was performed at three
ments of analysis. Being able to measure time points: immediately before treatment, im-
easily the effects of therapeutic experiences mediately after, and 24 hours later. Differential
can validate the benefits of intervention. In expression between EFT and control was found
order to test the feasibility of gene expres- in numerous genes implicated in overall health
sion testing in a private practice setting, this (p < 0.05). Further, the differentially expressed
study compared messenger ribonucleic acid genes in this study were shown to be linked
(mRNA) and gene expression before and after to immunity, pro or anti-inflammatory, as well
psychotherapy and a control condition. With as neuronal processes in the brain. Ten of the
four non-clinical adult participants, it piloted a 72 differentially expressed genes are identified
novel methodology using saliva stored at room as promising targets for downstream research.
temperature. A preliminary test of the inter- The data show promise for the future use of
leukin-8 (IL8) gene in both blood and saliva salivary samples to determine the effects of
was performed in order to determine equiva- therapy; this pilot protocol also illustrated the
lency in the two biofluids; convergent validity challenges and limitations of novel technolo-
was found. Following saliva test validation, a gies employed in biopsychology.
broad, genome-wide analysis was performed
to detect differential gene expression in sam- Keywords: epigenetics, DNA, mRNA, gene
ples collected before and after treatment with expression, protein synthesis, brain plasticity,
Emotional Freedom Techniques (EFT), an neurogenesis, biopsychology
Marjorie “Beth” Maharaj, PhD, a licensed mental health Psychology is a broad field with many
counselor with an interest in experimental psychology, procedures and schools of thought regard-
uses therapies such as EFT, EMDR, and mindfulness in
ing the treatment of mental and emotional
her private practice. Correspondence: 2609 SW 33rd St.,
problems. As the field broadens with ever-evolv-
Unit 103, Ocala, FL 34471; e-mail: beth@serenityocala.
ing eclecticism and fine-tuning of psychothera-
com. Acknowledgments: Appreciation and sincerest thanks
to Corina Guethlin, PhD, as my primary advisor; Garret peutic techniques and modalities, more questions
Yount, PhD, as my outside consultant for molecular biology; arise that pertain to the biological mechanisms
Kelli Barr, PhD, as my local molecular biology consultant; behind client recoveries and transformations
Nicolette Bovell as my research assistant; UT Southwestern
following treatment and self-maintenance. It is
Microarray Core Lab for sample processing and analysis; Elliot
theorized that any novel experience, including
Benjamin, PhD, and Christopher Johannes, PhD, HMD, as
experiential psychotherapeutic interventions,
my tertiary advisors. Disclosures: The author declares no
conflict of interest. can impact gene expression in humans, resulting
Differential Gene Expression after EFT Treatment Energy Psychology 8:1 • May 2016 17
in brain changes. This phenomenon is known as much shorter treatment time frames (Greenberg &
neurogenesis or brain plasticity (Siegel, 1995; Watson, 1998; Karatzias et al., 2011; de Roos et al.,
Kandel, 1998, 2001; Montag-Sallaz, Welzl, 2011; Church, 2013a, 2013c). These interventions
Kuhl, Montag, & Schachner, 1999; Rossi, 2002; typically include techniques that induce the relaxa-
Rutishauser, Mamelak, & Schuman, 2006). It has tion response (RR) to lower emotional distress,
been argued that effective psychotherapy may be anxiety, or insomnia. RR meditation, defined as a
viewed as an epigenetic intervention, regulating mind-body intervention, is known to offset the phys-
stress genes such as those that code for cortisol iological effects of stress (Benson & Klipper, 1975).
and epinephrine, as well as regulating the auto- Brain plasticity, or neurogenesis, is the lifelong
nomic nervous system (Feinstein & Church, 2010; ability of the brain to change, grow, and reorganize
Church 2013c). neural pathways based on new experiences and
The science behind the merging fields of even injury (Eriksson et al., 1998; Rossi, 2002).
biology and psychology has proliferated over the Genetic processes have been shown to result in neu-
last decade with researchers in both fields inte- ronal growth in the brain by increasing the number
grating aspects of the other (Rossi, 2002; Rossi, of synapses between neurons (Eriksson et al., 1998;
Rossi, Yount, Cozzolino, & Iannotti, 2006; Siegel, Kandel, 1998, 2001; Neville & Bavelier, 2000).
2012). Technologies have emerged in the fields of These are the processes responsible for neuronal
functional magnetic resonance imaging (fMRI; brain growth by way of genetic processes, also
Petrella, Mattay, & Doraiswamy, 2008), endocri- known as brain plasticity. Plasticity may be trig-
nology (Yehuda et al., 2009), and molecular biology gered by adverse life experiences, such as trauma,
(Yount, 2013) that permit the experimental testing loss, and injury. Plasticity may also be triggered by
of the effect that psychotherapy has on neurogen- positive experiences such as novelty, learning, and
esis (Eriksson et al., 1998; Ackerman, Martino, psychotherapeutic interventions. Therapies that
Heyman, Moyna, & Rabin, 1998; Montag-Sallaz employ this effect may therefore be regarded as
et al., 1998; Ramanan et al., 2005; Xiang et al., epigenetic interventions (Church, 2013c).
2008; Boyke, Driemeyer, Gaser, Büchel, & May, Experiences trigger protein synthesis medi-
2008). Recent gene expression research has enabled ated by messenger ribonucleic acid (mRNA),
investigators to study the effects of experiences resulting in a cascade of physiological, neuronal,
such as psychotherapeutic interventions. The notion and structural changes (Strachan & Read, 1999).
that environment and experience change the brain’s Combining the study of psychotherapy and the
neurological wiring has evolved from a hypothesis processes of neurogenesis is referred to as Inter-
into an empirically demonstrated reality (Anderson personal Neurobiology (Siegel, 2012). Empirical
et al., 2004; Erk et al., 2010; Hölzel et al., 2011). investigation in this area of study has been chal-
lenged due to the lack of a noninvasive method of
sample collection. The validation of a biofluid col-
Psychotherapy and Neuroplasticity
lection protocol would allow the measurement of
Psychotherapeutic modalities are broadly gene expression and the exploration of the effects
eff ec tive, with no one method showing clear supe- of psychotherapy as an epigenetic intervention.
riority over others (Wampold, Mondin, Moody,
Stich, Benson, & Ahn, 1997; Ahn & Wampold,
Epigenetics and Neuroplasticity
2001). Psychotherapy is also efficacious for physi-
cal conditions, with a great deal of evidence sup- Genes are the mechanisms by which living
porting the link between mental and physical health organisms inherit features from their ancestors.
(Alexander, Arnkoff, & Glass, 2010; Church, The genotype is the genetic makeup of a cell, an
2013c). The American Psychological Association organism, or an individual. The cell interprets
(APA) recognizes the benefits and effectiveness of the genetic code stored in DNA when the gene is
psychotherapy, and suggests that psychotherapy expressed, and the properties of that expression
should continue to be included within the primary give rise to the organism’s phenotype and observ-
health care system. Though evidence supports able characteristics, including behavior.
equivalency and comparability of psychotherapeu- Gene expression can be influenced by envi-
tic interventions, experiential and somatic thera- ronment and experience resulting in phenotypic
pies have been shown to yield improvements in changes such as neurogenesis. mRNA is an
18 Energy Psychology 8:1 • May 2016 Differential Gene Expression after EFT Treatment
of stress and preserve telomere length, mitigating
the aging process. Telomere length has been cor-
related with age-related health decline as well as
how health is negatively or positively affected by
the environment, stressful experiences, and medita-
tion (Kotrschal, Ilmonen, & Penn, 2007; Okereke
et al., 2012; Ladwig et al., 2013; Epel, 2009; Epel,
Daubenmier, Moskowitz, Folkman, & Blackburn,
2009; Jacobs et al., 2011).
Figure 1. The blueprint in DNA for the synthesis of a
protein is mediated by mRNA. Emotional Freedom Techniques (EFT)
Emotional Freedom Techniques (EFT) is an
information carrier that codes for the synthesis of evidence-based psychotherapy self-help technique.
one or more proteins. Proteins can be synthesized It has been validated in over 100 studies, meta-
using the information in mRNA as a template analyses, and review papers accessible via an online
(Figure 1). Downstream genes can be upregulated bibliography (Research.EFTuniverse.com). EFT
or downregulated, turned on or off by messenger uses elements of exposure and cognitive therapies,
proteins. and combines them with acupressure (i.e., fingertip
Many factors determine whether a gene is on stimulation of acupuncture points). It is described in
or off, such as the time of day, whether or not the a manual that has been available since the inception
cell is actively dividing, its local environment, and of the method, leading to its uniform application in
chemical signals from other cells. Upregulation research, training and certification (Craig & Fowlie,
and downregulation of genes affects the very wir- 1995; Church, 2013b). Studies have demonstrated
ing of the brain and body, predisposing the body its efficacy for a wide variety of psychological con-
toward the development of disease, or improving ditions and physical symptoms (Wells, Polglase,
health, thinking, and memory (Church, 2013c; Andrews, Carrington, & Baker, 2003; Brattberg,
Montag-Sallaz et al., 1999; Ramanan et al., 2005; 2008; Karatzias et al., 2011; Church, De Asis, &
Pfenning, Schwartz, & Barth, 2007; Yehuda et al., Brooks, 2012; Church, Yount, & Brooks, 2012).
2009; Brocke et al., 2010). Meta-analyses have found “large” treatment
Information transfer between DNA, RNA effects for anxiety, depression, and PTSD (Clond,
(both nucleic acids), and protein is multidimen- 2016; Nelms & Castel, 2016; Sebastian & Nelms,
sional and occurs in several different ways. There 2016). The treatment time frames described in
are direct transfers of information between DNA, these reviews were brief, ranging from one session
RNA, and proteins. DNA can be copied to DNA for phobias to between four and 10 sessions for
(replication). DNA information can be copied into PTSD. The treatment effects of EFT were found to
mRNA (transcription). mRNA then carries a copy extend over time. Systematic review papers have
of DNA to other DNA, binding to it and triggering also described the efficacy of EFT for pain, trau-
its expression (gene expression). In general, gene matic brain injury, sports performance, fibromyal-
expression is regulated through changes in the num- gia, and other physical conditions (Church, 2013b;
ber and type of interactions between proteins that Feinstein, 2012; Feinstein & Church, 2010). The
collectively influence the transcription of DNA and effect sizes for EFT found in meta-anlyses are
the translation of RNA (Strachan & Read, 1999). larger than those typically observed in conventional
Telomeres are the molecular tails of DNA psychotherapy and psychopharmacology trials
strands. Each time DNA replicates, a pair of tel- (Clond, 2016; Nelms & Castel, 2016; Sebastian &
omerase molecules is lost (Sprung, Sabatier, & Nelms, 2016). Several dismantling studies have
Murnane, 1996; Ning et al., 2003). Telomere isolated the acupressure component of EFT from
tails shorten at a stable rate of about 1% per year, the conventional cognitive and exposure protocols
and are regarded as the most accurate biologi- that EFT shares with other therapeutic methods
cal marker of aging (Church, 2013c). Positive (reviewed in Church & Nelms, 2016). They find
changes in lifestyle, such as meditation and a that the acupuncture point stimulation element
healthy diet, can ameliorate the oxidative effects of EFT is an active ingredient and not simply an
Differential Gene Expression after EFT Treatment Energy Psychology 8:1 • May 2016 19
2003). Further, saliva mRNA samples degraded too
quickly (Hu et al., 2008).
More recently, it has been established that
saliva as a biological sample has the potential,
as an easily collected body fluid, for human gene
expression and experience research (Zubakov,
Hanekamp, Kokshoorn, van Ijcken, & Kayser,
2008; Zubakov, Boersma, Choi, van Kuijk, &
Wiemer, 2010). Technology now exists that allows
for saliva collection from participants before and
Figure 2. Acupressure points prescribed in The EFT after psychotherapeutic intervention by way of
Manual (Church, 2013b). in-vial mRNA purification. Storage, analysis, and
comparison can be accomplished at room tem-
inert placebo. This is confirmed by studies using perature rather than requiring frozen samples.
fMRI and other biological measures to investigate The study used the Oragene saliva self-collection
the brain’s response to acupuncture; all show kit (OrageneRNA for Expression Analysis Self-
regulation of the brain regions and brain-wave fre- Collection Kit, dnaGenotek, Ontario, Canada).
quencies associated with fear (Dhond, Yeh, Park, The Oragene device consists of a proprietary fluid
Kettner, & Napadow, 2008; Bai et al., 2009, 2010; matrix in which samples are stored. This device
Witzel et al., 2011; Liu et al., 2011). EFT has also is most commonly used in the medical and pub-
been shown to regulate cortisol (Church, Yount, & lic health sectors for downstream isolation of
Brooks, 2012). A study of the epigenetic effects of genomic DNA. The manufacturer’s directions are
EFT in veterans with PTSD found regulation of six easy to follow; the stabilizing liquid is inside the
genes including those in the interleukin family that lid of the vial. For the saliva sample, the partici-
are linked to the stress response (Church, Yount, pant spits into the tube, the lid is tightened, which
Rachlin, Fox, & Nelms, 2015). releases the stabilizing liquid, and then gentle
Besides its cognitive, exposure, and acupres- shaking mixes the stabilizer with the saliva.
sure tapping components, EFT uses a bilateral The Oragene kit is to date the only all-in-
brain activation strategy called the 9 Gamut Pro- one system for the collection, stabilization, and
cedure that is hypothesized to increase communi- transportation of high-quality mRNA from saliva
cation between the right and left hemispheres of ( Figure 3). This product literature claims that it
the brain through the corpus callosum. Before and yields “high quality total RNA.” It is advertised as
after the application of EFT, clients self-assess a noninvasive and easy-to-use self-collection tube
their degree of stress on an 11-point Likert scale. that remains stable for months at room tempera-
They then use EFT’s “Basic Recipe” of acupoint ture; therefore, no sample freezing is necessary.
stimulation while vividly recalling a traumatic With recent advances in the field of molecu-
event. This is followed by the 9 Gamut, then by a lar biology, it is possible to scan the entire genome
second application of the Basic Recipe (Figure 2). for gene expression using a more cost effective
high-throughput, multiplexed bead-based tech-
nology (Yang, Tran, & Wang, 2001; Krutzik &
The Viability of Saliva Sampling
Previously in gene expression research, blood
samples were needed for profiling and analyses.
Easily collected fluids such as saliva were dismissed
due to several inadequacies. Saliva was considered
to have too much extraneous DNA from viruses
and bacteria to discriminate human gene expression
(Kumar, Hurteau, & Spivack, 2006; Chiappelli,
Iribarren, & Prolo, 2006). Before advances in puri-
fication and amplification technology, the quantity
of mRNA obtainable from saliva was insufficient
to measure significant changes (Bartlett & Stirling, Figure 3. Saliva sample collection vial.
20 Energy Psychology 8:1 • May 2016 Differential Gene Expression after EFT Treatment
Nolan, 2003; Elshal & McCoy, 2006; Jacobson, Preliminary Proof of Methods
Oliver, Weiss, & Kettman, 2006; Bruse, Moreau, Validation Test
Azaro, Zimmerman, & Brzustowicz, 2008; Leng,
No prior study collecting sufficient quantities
McElhaney, Walston, Xie, Fedarko, & Kuchel, 2008).
of mRNA from samples stored in a preservation
Prior to these advances, it was necessary to look at
matrix at room temperature has been published in
a very narrow selection of genes, sometimes only
the literature. It was necessary to determine whether
one at a time using RT-PCR (Real-Time polymerase
the mRNA level for a gene in saliva samples was
chain reaction) technology, which is very costly.
comparable to the level in blood samples in order
The current study sought to elucidate the effect
to demonstrate the feasibility of the planned study.
of psychotherapy on gene expression by measur-
Accordingly, a preliminary validation of the Ora-
ing expression before and after a single session of
gene device was performed. This preliminary
EFT. The recent availability of noninvasive saliva
investigation tested logistics and procedure feasi-
tests offers the possibility of elucidating how
bility before the launch of the planned study.
psychotherapy works as an epigenetic influence
For this validation, samples of blood and saliva
on gene expression. The aim of this study was to
were collected before, after, 2 hours after, 4 hours
observe the genetic response of healthy individu-
after, and 6 hours after a novel experiential psycho-
als to a psychotherapeutic intervention using saliva
therapeutic intervention and stored at room temper-
sample collection. A second objective was to inves-
ature for 2 weeks. mRNA was then extracted from
tigate the feasibility of using saliva rather than blood
the samples according to manufacturer recommen-
as a biofluid suitable for conducting genetic research
dations. mRNA expression from blood and saliva
in a clinical setting, and delineate the parameters of
was quantified for the interleukin-8 (IL8) gene,
a protocol to ensure successful data collection.
a pro-inflammatory gene (Shahzad et al., 2010),
from the samples using Real-Time PCR (RT-PCR).
Method IL8 was one of the genes found to be significantly
regulated by EFT in the prior gene expression study
Participants and Blinding
using blood samples analyzed with PCR (Church,
The study was approved by the Institutional
Yount, Rachlin, Fox, & Nelms, 2016).
Review Board (IRB) of Akamai University. Poten-
The preliminary validation test found that both
tial participants were English-speaking adults
saliva and blood were sensitive biofluids, exhibit-
aged 18 to 65. Prior to enrollment, they were
ing significant upregulation, relative to a twofold
screened to verify nonclinical mental health status,
change threshold, of the IL8 gene from baseline
and excluded if they scored above 20 on the Brief
(Figure 4). The Cycle threshold (Ct value) is the
Symptom Inventory 18 (BSI 18; Derogatis, 2001),
number of cycles of PCR amplification at which
adapted for the purpose of this study to include
the signal of the target gene exceeds background
“lifetime” instead of just the last 7 days. Of 24
noise (Shiao, 2003). mRNA encoding the IL8 gene
potential participants screened, 10 were excluded
extracted from saliva was detected at a Ct of 22.
on this basis. Of the remaining 14, five were
selected based on their availability for the follow-
ing two weekends. All provided informed consent.
All study data were de-identified and coded
to protect the participants’ identities and facilitate
impartial analysis of the samples. One participant
was disqualified due to admission of a psychiat-
ric diagnosis leaving a final N = 4. Sample vials
were labeled with five-digit identifying codes
determined by a random number generator (http://
stattrek.com/statistics/random-number-generator.
aspx), thus effectively blin ding the molecular
Figure 4. RT-PCR Ct values for IL8 gene expression of
biology analysis. The key was not provided to the
blood versus saliva. Time 0, BI = before intervention;
molecular biology team until after the samples Time 1, AI = after intervention; Time 2, AI = 2 hours after
were processed for mRNA extraction, gene detec- intervention; Time 3, AI = 4 hours after intervention;
tion, and gene expression. Time 4, AI = 6 hours after intervention.
Differential Gene Expression after EFT Treatment Energy Psychology 8:1 • May 2016 21
IL8 mRNA from blood was detected at a Ct of 24. sample, 50 ng of total RNA was labeled with biotin
Lower Ct values indicate greater quantities of and then hybridized using the Illumina chip. The
mRNA. It is expected for biofluids to have different chips were then scanned by an Illumina HiScan-
expression patterns for a specific gene as a result SQ scanner. The level of hybridization was meas-
of their differing constituents. The similarity of the ured via Cy3-Streptavidin fluorescence. Data were
results obtained from the two biofluids indicated the normalized to background then analyzed using
feasibility of using saliva for the planned study, and Illumina Genomestudio software (San Diego,
demonstrated the logistical feasibility of measuring CA). This platform was chosen for this study as it
differential gene expression before and after EFT. allowed for the detection of specific gene expres-
sion activity across the entire human genome at
a fraction of the cost of using the RT-PCR proce-
Sample Collection and Analysis
dure, yet with comparable detection capabilities.
After determining that saliva was a viable bio-
fluid, the main study proceeded. Saliva samples
were first collected under control conditions, and Statistical Analysis
then, a week later, from the same participants
Using the Illumina Genomestudio software,
before and after EFT. The control was 50 minutes
gene expression data were pooled and compared
of non-therapeutic conversation moderated by a
across each condition and time point. Data from
non-therapist research assistant. The experimental
individual participants were normalized using
condition was 50 minutes of EFT.
quantile normalization. This is a method used
Study participants provided 1 ml of saliva by
to make the distribution, median, and mean of
expectorating directly into the Oragene collection
probe intensities the same for every sample. The
vial. Saliva samples were collected from all par-
normalization distribution is chosen by aver-
ticipants in both groups immediately before (T0),
aging each quantile across samples in order to
immediately after (T1), 4 hours after (T2), and
generate an average signal (AVG-Signal). The
24 hours after (T3) the 50-minute treatment. A
data analysis included the p value and other
total of 40 samples were collected one week apart
descriptive statistics such as Standard Deviation,
for control and experimental conditions. After col-
Standard Error, and t-test for each gene. The
lection, vials were sealed and stored at room tem-
p value indicates the statistical significance that
perature until RNA extraction.
the detected signal on the chip was differentially
As these are novel methods, it was decided that
illuminated when compared to the controls and
an initial trial of 12 samples on one chip would be
background noise. Detection p value is a sta-
attempted first. This was performed at the Univer-
tistical calculation that provides the probability
sity of Texas Southwestern Microarray Core Lab.
that the signal from a given probe is greater than
The first chip was processed 35 (+ or – 7 days) days
the average signal from the negative controls.
after collection. By the time these results had been
A p value < 0.01 indicates that a specific gene
received and it was determined that quality mRNA
exhibited significant up- or downregulation from
had been quantified, only 12 of the next 24 samples
controls and background noise.
passed quality controls. Extraction of the second
Genes that were differentially expressed
chip occurred about 80 days after sample collec-
between groups were identified by comparing
tion. dnaGenotek was contacted about the degrada-
the expression values of the genes. Differential
tion of the samples, which their literature confirmed
expression was determined via Student’s t-test
could be “stored at room temperature for months,”
for the treatment group in question. Differences
and then stated that the expiration point of their col-
between conditions (CC = control; EC = experi-
lection vials should be considered 60 days.
mental) and time point with p < 0.05 were consid-
ered significant.
Labeling, Hybridization, and Data
Of the 40 samples collected, only 24 samples
Analysis were suitable for analysis based on extractable
The Illumina Human HT-12-V4 BeadChip mRNA. Obtaining high quality mRNA was prob-
array is made up of randomly positioned silica lematic due to sample degradation. There was var-
beads, each containing hundreds of thousands of iability in the number of detectable genes found in
copies of a specific probe sequence. From each each sample, displayed in Table 1. This was most
22 Energy Psychology 8:1 • May 2016 Differential Gene Expression after EFT Treatment
Table 1. Number of Genes Detected in Each Sample time point and condition. The data from Time 2
per Condition and Time Point out of 47,000 Genes (T2), 4 hours after the intervention, were elimi-
nated from further analysis due to too few usable
Sample groups CS ES
samples to compare.
T0 CS5T0: 6410 ES5T0: 416*
CS4T0: 522*
Differential Gene Expression
CS3T0: 2781 ES4T0: 618*
Differential gene expression was measured
CS2T0: 1585
using the methods outlined previously. Because of
CS1T0: 425* ES3T0: 3434
the size and magnitude of the data generated (out
T1 CS5T0: 8548 ES5T1: 701* of 47,000 genes), only 10 of the 72 differentially
CS3T0: 4728 ES2T1: 9064 expressed genes will be mentioned for the results
CS2T1: 1504 ES4T1: 618* (Figure 5, Table 3). Downstream investigation is
suggested for these 10 differentially expressed
T2 CS5T2: 10634 ES5T2: 9064
genes: CCNB1IP1 is also known as Cylin B1 inter-
ES3T2: 516*
acting protein. It is involved with the progression
T3 CS5T3: 7741 ES5T3: 570* of the cell cycle. CCNB1IP1 showed significant
ES4T3: 521* upregulation, 8-fold, immediately after EFT com-
CS4T3: 709* ES3T3: 427* pared to the control condition (p < 0.02). Expression
of CCNB1IP1 has been linked to tumor suppression
CS3T3: 1267 ES2T3: 1576
(Ma et al., 2013). COPS7A is a subunit of COP9
Note: *These samples have a very low number of detectable and is known as constitutive photomorphogenic
genes. signalosome subunit 7A. COP9 and its subunits play
a role in protecting and repairing damaged DNA due
likely due to the time lapse between collection and to UV radiation (Füzesi-Levi et al., 2014). COPS7A
sample processing. showed significant upregulation, 5-fold, after EFT
Samples obtained at Time 2, the time point compared to control condition (p < 0.02). DAB2,
4 hours after the intervention, did not yield Disabled-2, is a FOXP3 target gene for regulating
enough usable mRNA to be compared across
conditions. The data for this time point have
Table 2. Sample Information
therefore been eliminated from the results due
to the poor quality mRNA extraction from most Sample Samples in CS Samples in ES
of the samples. Participants were instructed on groups group group
study procedures and collection times; however,
T0 5 CS5T0 3 ES5T0
instructions may not have been followed, as this
CS4T0
was the only time point that was not observed by
the research assistant. It is possible the partici- CS3T0 ES4T0
pants ate, smoked, or chewed gum before collect- CS2T0
ing that particular sample. CS1T0 ES3T0
T1 3 CS5T0 3 ES5T1
Results CS3T0 ES2T1
CS2T1 ES4T1
RNA Extraction and Validation
T2 1 CS5T2 2 ES5T2
Samples were extracted following the steps
listed in the Oragene purification protocol using ES3T2
TRIzol LS reagent (Invitrogen, Carlsbad, CA). T3 3 CS5T3 4 ES5T3
Total RNA samples (1 ml) were analyzed for
ES4T3
quality and purity by chip electrophoresis using
CS4T3 ES3T3
Agilent’s 2100 Bioanalyzer (Agilent Technolo-
CS3T3 ES2T3
gies, Santa Clara, CA) and reagents from the RNA
6000 pico kit. Table 2 shows the number of sam-
Note: Total 24 out of 36 samples were processed on Illumina
ples that passed extraction and validation for each Human HT-12 arrays.
Differential Gene Expression after EFT Treatment Energy Psychology 8:1 • May 2016 23
Figure 5. Differential gene expression of 10 genes of interest and basic function: Average signal (AVG_SIGNAL)
of control condition (CC) compared to experimental condition (EC).
T-cell function. DAB2 has tumor suppression and (p < 0.02). PDE4DIP, phosphodiesterase 4D
anti-cancer effects. Loss of DAB2 expression in interacting protein has implications for mood,
breast cancer can be detrimental to prognosis (Xu, memory, and learning (Kim, Cho, Lee, & Web-
Zhu, & Wu, 2014). Downregulation of DAB2 ster, 2012; Shapshak, 2012). PDE4DIP upregulated
switches TGF-B (tumor growth factor-B) from a 2-fold immediately after EFT (p < 0.02). MCL1,
tumor suppressor to a tumor promoter (Hannigan Myeloid Cell Leukemia 1, mediates apoptosis (pro-
et al., 2010). DAB2 showed significant upregula- grammed cell death). MCL1 is named and known
tion, 3-fold, after EFT compared to control condition for the role it plays in cancer promotion when it is
24 Energy Psychology 8:1 • May 2016 Differential Gene Expression after EFT Treatment
Table 3. Summary of 10 Genes and Translational Functions
Gene Discovered function(s) References
CCNB1IP1 Tumor suppression Ma et al., 2013
COPS7A Protects against UV radiation Groisman et al., 2003;
Füzesi-Levi et al., 2014
DAB2 Cancer tumor suppression Tong et al., 2010;
Hannigan et al., 2010;
Xu, Zhu, & Wu, 2014
PDE4DIP Implications for mood, memory, and learning Kim, Cho, Lee, & Webster, 2012;
Shapshak, 2012
MCL1 Neuronal survival after DNA damage. Xingyong et al., 2013;
Suppression, cancer prevention Lestini et al., 2009
PSMB1 Increases type 2 diabetes insulin resistance. Yamauchi et al., 2013;
Upregulation related to anticancer Keutgens et al., 2010
PTMA Mediates immune function by increasing resistance Su et al., 2013;
to opportunistic infections. Bowick et al., 2010
Antiviral properties when upregulated
PXMP4 Cancer tumor suppression Zhang et al., 2010
ARL6IP4 Resistance and recovery from emotional stress Wu et al., 2013; Carhuatanta,
and antiviral activity Shea, Herman, & Jankord, 2014
SEZ6 Enhances synaptic connectivity in the brain by Gunnersen et al., 2007
promoting dendritic arborization (branching) of neurons
overexpressed (Lestini et al., 2009; Ertel, Nguyen, Shea, Herman, & Jankord, 2014). ARL6IP4 upreg-
Roulston, & Shore, 2013). With moderate expres- ulated 2-fold after EFT (p < 0.002). SEZ6, seizure
sion, however, MCL1 has been found to help neu- related gene 6, has implications for enhancing syn-
ronal survival after DNA damage (Xingyong et al., aptic connectivity in the brain by promoting den-
2013). MCL1 showed significant upregulation after dritic arborization, (branching) of neurons (Gun-
EFT compared to control condition (p < 0.004). nersen et al., 2007). SEZ6 showed significant
PSMB1, Protaesome subunit beta type-1, is a mul- upregulation, 3-fold, after EFT compared to control
ticatalytic proteinase complex with a highly ordered (p < 0.05). A basic PubMed search on 32 of the 72
ring-shaped 20S core structure. PSMB1 is known to genes differentially expressed in the immediately
play a role in insulin resistance when overexpressed after EFT condition compared to control condition
(Yamauchi, Sekiguchi, Shirai, Yamada, & Ishimi, yielded some very interesting possibilities for how
2013). PSMB1 remained constant throughout the EFT might affect immunity and inflammation sys-
experiment, unlike the control condition, which was temically (Table 4). Also immediately after EFT,
variable (p < 0.002). PTMA, Prothymosin alpha, genes were expressed that are known to code for
mediates immune function by increasing resistance structural neurogenesis and brain plasticity.
to opportunist infections. PTMA showed significant
upregulation, 5-fold, after EFT compared to control
Discussion
condition (p < 0.04). PXMP4, Peroxisomal Intrin-
sic Membrane Protein, has been found to have can- This study piloted a novel methodology
cer tumor suppression properties when expressed of using saliva to measure mRNA and gene
(Zhang et al., 2010). PXMP4 showed significant expression before and after therapy in order to
upregulation, 5-fold, after EFT compared to control test the feasibility of using gene expression to
condition (p < 0.04). ARL6IP4, ADP ribosylation examine the physiological correlates of effective
factor like GTPase 6 interacting protein 4, is known treatment. A broad, genome-wide analysis was
for resistance and recovery from emotional stress performed to detect differential gene expression
and antiviral activity (Wu et al. 2013; Carhuatanta, from saliva samples collected before and after
Differential Gene Expression after EFT Treatment Energy Psychology 8:1 • May 2016 25
s
e
m
Gene function Oral cavity related Cancer tumor regulation Hormone related Energy & metabolism related DNA methylation; relates to brain function & behavior Encodes an epigenetic regulator promoting transcriptional plasticity Cancer tumor suppressor Memory & learning Mediates membrane sculpting One of the most important antioxidant enzyin humans Prevents UV damage Tumor suppressor Cancer promotion/autism & brain damage Neuronal survival after DNA damage Enhances brain synapse connectivity Tumor suppressor
al
v
p
1.diff 66234 31552 69039 71318 99931 79955 02478 35445 92237 20716 2078 81662 13861 46044 47686 58648
T 0 1 1 2 5 6 7 7 7 8 3 3 5 5 9 9
S- 01 01 01 01 01 01 01 01 01 01 02 02 03 04 04 04
E 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.
Symbol SLK RPL19P9* EFCAB6 UCP3 PPM1G KDM6B DAB2* PDE4DIP* PACSIN2 GPX1 COPS7A* CCNB1IP1* SLC25A24* MCL1* SEZ6* PXMP4*
on d
Genes Differentially Expressed after EFT Gene function T cell/immunity Growth/immunity Antiviral activity Insulin regulation Brain white matter regulator Cellular response to external stimuli Blood cells development & differentiati CNS Brain pituitary function Saliva related Male fertility Cell recognition in CNS Brain synapse shape, implicated in moochanges Regulation of stress response Inflammation & immune response Nicotinic pathway signaling ditions and time points.
n
ons of 32 of the 72 ES-T1.diff pval 0.000140196 0.000726317 0.002220663 0.002253853 0.002633457 0.003499107 0.003522641 0.004008878 0.004695438 0.006684133 0.007274315 0.008081845 0.008235929 0.008412043 0.009469124 0.009792715 G signal across all co
cti AV
Table 4. Fun Symbol MAL PVRL3 ARL6IP4* PSMB1* EIF2B2 DGKD SBDS* FLJ45337* GNAI3 PRB3 RFX2 CNTNAP3* MEGF10 LSM1 CASP1 NAPRT1 Note: *Stable
26 Energy Psychology 8:1 • May 2016 Differential Gene Expression after EFT Treatment