Research Article Validation of Reference Genes for ...downloads.hindawi.com/journals/bmri/2014/198582.pdfBioMed Research International T : Stability of genes candidates to normalize

Research ArticleValidation of Reference Genes for Normalization GeneExpression in Reverse Transcription Quantitative PCR inHuman Normal Thyroid and Goiter Tissue

Raquel Weber1 Ana Paula Santin Bertoni12 Laura Walter Bessestil3 Beatriz Maria deAzevedo Assis Brasil4 llma Simoni Brum3 and Tania Weber Furlanetto1

1 Programa de Pos Graduacao emMedicina CienciasMedicas Universidade Federal do Rio Grande do Sul Rua Ramiro Barcelos 240090035-903 Porto Alegre RS Brazil

2 Programa de Pos Graduacao em Ciencias da Saude Universidade Federal de Ciencias da Saude Porto Alegre Rua Sarmento Leite245 90050-170 Porto Alegre RS Brazil

3 Departamento de Fisiologia Instituto de Ciencias Basicas da Saude Universidade Federal do Rio Grande do Sul Rua Sarmento Leite500 90050-170 Porto Alegre RS Brazil

4 Servico de Patologia Hospital de Clınicas de Porto Alegre Rua Ramiro Barcelos 2350 90035-903 Porto Alegre RS Brazil

Correspondence should be addressed to Tania Weber Furlanetto taniafurlanettogmailcom

Received 28 February 2014 Accepted 18 April 2014 Published 11 May 2014

Academic Editor Muy-Teck Teh

Copyright copy 2014 Raquel Weber et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate methodfor quantifying mRNA transcripts but normalization of samples is a prerequisite for correct data interpretation So thisstudy aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues Beta-actin(ACTB) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) succinate dehydrogenase subunit A flavoprotein (Fp) (SDHA)hypoxanthine phosphoribosyltransferase I (HPRTI) tyrosine 3-monooxygenasetryptophan 5-monooxygenase activation proteinzeta polypeptide (YWHAZ) and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goitertissues) by RT-qPCRThemeanCq and themaximum fold change (MFC) andNormFinder softwarewere used to assess the stabilityof the genes As a result ACTB gene wasmore stable thanGAPDH SDHAHPRTI YWHAZ and B2M In conclusion ACTB couldbe used to normalize RT-qPCR data in normal thyroid and goiter tissues

1 Introduction

Reverse transcription quantitative polymerase chain reaction(RT-qPCR) has been recognized as the most accurate sen-sitive and easy method for quantifying mRNA transcriptsin biological samples [1 2] Nevertheless normalization ofsamples is a prerequisite for correct data interpretation sinceits accuracy is significantly affected by sample quality reagentand operator technique [3]

Cell number quantification of rRNA or total RNA andreference genes are some standardization methods that havebeen proposed so far [2 4 5] Normalization with anothergene which is assumed to have stable expression is themethod of choice because this reference gene is exposed to

the same preparation steps as the gene of interest [6] There-fore a full procedure control is obtained during the wholeassay

A good reference gene needs to present low variation in itsexpression in the different samples which would be evaluatedand demonstrated to be minimally regulated during theindividual experiment or pathological condition Thereforethere is a need to validate genes to allow the accuracy of RNAtranscription analysis [3 6] Nowadays there is emergingevidence that reference genes could vary their expression indifferent cellular development stages and in other conditions[5 7 8]

The thyroid normal gland is a fairly homogenous struc-ture but in goiter a genetic heterogeneity is described in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 198582 5 pageshttpdxdoiorg1011552014198582

2 BioMed Research International

Table 1 Stability of genes candidates to normalize RT-qPCR for thyroid derived biological material

Study Material Reference gene stability MethodSantin et al 2013 [9] Human normal cells ACTBgtTBPgtGAPDHgtB2M NormFinderChantawibul et al 2012 [10] Human tissueslowast GAPDHgtACTBgtB2MgtRPLIAgtHPRT1 NormFinder

Lecchi et al 2012 [11] Bovine tissues including thyroid SF3A1=HBMSgtACTB geNormHMBSgtACTBgtGAPDH NormFinder

Li et al 2011 [12] Porcine thyroid tissue 18S rRNAgtUbiquitingtHistone H3gtACTB geNorm18S rRNAgtUbiquitingtHistone H3gtACTBgtGAPDH NormFinder

Lisowski et al 2008 [13] Bovine thyroid tissue TBP=HPRT1gtYWHAZgtACTB geNormlowast13 goiters 6 adenomas 4 carcinomas (2 follicular + 2 papillary) and 2 lymphocytic thyroiditis

follicular cells some nodules have a polyclonal origin andothers a monoclonal origin These changes may be related tomutations in oncogenes which do not produce malignancyper se but would alter growth and function [14 15] Thesealterations could modify the expression of many thyroidgenes including the ones usually described as referencegenes so the evaluation of candidates to normalize RT-qPCRis necessary

Little is known about the expression stability of referencegenes in thyroid cells or tissue as shown in Table 1

Considering the species there are many differences inreference genes stability evaluated in these studies Althoughin human tissues the same software was used to analyzedata differences in RT-qPCR and cells status could havecontributed to these findings In addition a stable expressionof a reference gene in one tumor type does not predict a stableexpression in another tumor type [7] In this way there is nouniversal reference gene being necessary to validate potentialreference genes for any experimental condition So this studyaimed to evaluate themost stable reference gene for RT-qPCRin human normal thyroid and goiter tissues

2 Material and Methods

21 Tissue Acquisition Normal thyroid and goiter tissueswere obtained from patients who underwent total thyroidec-tomy as part of treatment for differentiated thyroid cancerin the Hospital de Clınicas de Porto Alegre (HCPA) Afterthe evaluation of macroscopic and frozen sections of surgicalspecimens by two pathologists samples of normal thyroidor goiter tissue were frozen in liquid nitrogen and stored atminus80∘C until further processing The pattern used to confirmthe presence of goiter was a well-defined fibrous capsulewith a mixture of macrofollicles and microfollicles and insome cases some degenerative changes such as fibrosis andhemorrhage [16] This study was approved by the EthicsCommittee of HCPA (GPPG 12-0272)

22 Nucleic Acid Extraction and Reverse Transcription Nor-mal thyroid and goiter tissue were homogenized mechan-ically with Omnimix for 30 seconds in Trizol (InvitrogenLife Technologies Carlsbad CA USA) and total RNAwas extracted with the same commercial kit according tothe manufacturerrsquos protocol The RNA concentration and

purity were assessed with the NanoDrop 1000 Spectropho-tometer (Thermo Fisher Scientific Wilmington DE USA)Samples were then stored at minus80∘C 1 120583g total RNA wasreverse-transcribed to produce cDNA using random andoligo-dT primers and Superscript II reverse transcriptase(Invitrogen Life Technologies) in total reaction volumes of20120583L All cDNA samples were diluted 10-fold with diethylpyrocarbonate- (DEPC-) treated water and stored at minus20∘C

23 Gene Selection and Quantitative PCR Six genes com-monly used as reference in RT-qPCR gene expression exper-iments were selected (Table 1) beta-actin (ACTB related tocell structure) glyceraldehyde-3-phosphate dehydrogenase(GAPDH related to carbohydrate metabolism) succinatedehydrogenase subunit A flavoprotein (Fp) (SDHA relatedto energy metabolism) hypoxanthine phosphoribosyltrans-ferase I (HPRTI related to nucleotide metabolism) tyrosine3-monooxygenasetryptophan 5-monooxygenase activationprotein zeta polypeptide (YWHAZ related to cell growthand death) and beta-2-microglobulin (B2M related tomajorhistocompatibility complex) Primers sequence shown inTable 2 was described previously [17] Quantification ofamplified samples was performed based on a standard curvewith five successive tenfold dilution points of a pool of cDNAsamples

RT-qPCR was performed using Applied BiosystemsStepOne Real-Time PCR System using Kit Platinum SYBRGreen qPCR SuperMix-UDG (Invitrogen Life TechnologiesCarlsbad CA USA) The RT-qPCR was performed with aninitial denaturation (10min at 95∘C) followed by 40 cyclesof denaturation (15 s at 95∘C) annealing and extension (45 sat 60∘C) Melting curve was acquired to ensure that a singleproduct was amplified in each reaction

24 Statistics To evaluate the stability of the candidatereference genes in thyroid normal and goiter tissues theNormFinder algorithm was used [18] In addition the analy-sis of raw quantification cycle (Cq) values of each gene wasused to evaluate their stability Mean Cq values standarddeviation (SD) coefficient of variation (CV) and maximumfold change (MFC the ratio of the maximum and minimumvalues observed within the dataset) were calculated

BioMed Research International 3

Table 2 Primers sequence product length and NCBI reference sequence for reference genes by RT-qPCR

Genesymbol Name Forward and reverse primers Product

length (bp)NCBI reference

sequence

ACTB Beta-actin 51015840-CTGGAACGGTGAAGGTGACA-31015840 140 NM 001101351015840-AAGGGACTTCCTGTAACAATGCA-31015840

B2M Beta-2-microglobulin 51015840-CTATCCAGCGTACTCCAAAG-31015840 165 NM 004048251015840-ACAAGTCTGAATGCTCCACT-31015840

GAPDH Glyceraldehyde-3-phosphatedehydrogenase

51015840-CTTTGTCAAGCTCATTTCCTGG-31015840 133 NM 002046351015840-TCTTCCTCTTGTGCTCTTGC-31015840

HPRT1 Hypoxanthine phosphoribosyltransferase 1 51015840-AGATGGTCAAGGTCGCAAG-31015840 128 NM 000194251015840-GTATTCATTATAGTCAAGGGCATATCC-31015840

SDHA Succinate dehydrogenase complex subunitA flavoprotein (Fp)

51015840-TGGTTGTCTTTGGTCGGG-31015840 85 NM 004168251015840-GCGTTTGGTTTAATTGGAGGG-31015840

YWHAZTyrosine 3-monooxygenasetryptophan5-monooxygenase activation protein zetapolypeptide

51015840-CAACACATCCTATCAGACTGGG-3101584051015840-AATGTATCAAGTTCAGCAATGGC-31015840 133 NM 0011356991

bp base pair Modified from Souza et al 2012 [17]

ACTB B2M GAPDH HPRT1 SDHA YWHAZACTB B2M GAPDH HPRT1 SDHA YWHAZ

Intr

agro

up v

aria

tion

GoiterNormal

GoiterNormal

Inte

rgro

up v

aria

tion

0150

0100

0050

0000

minus0050

minus0100

minus0150

900

800

700

600

500

400

300

200

100

000

Figure 1 Intra- and intergroup variation of six candidate reference genes in goiter and normal thyroid tissues samples

3 Results

Six reference genes were amplified in 14 thyroid samples 7normal and 7 goiter All RT-qPCR assays produced a singlepeak in the melting curveThemean Cq cycle values SD CVandMFCobtained for each gene in goiter and normal thyroidare described in Table 3 Analyzing these data none of thegenes had a constant expression Moreover B2M showedhigher variability in Cq values Thereby we proceeded theanalysis of quantification data using the NormFinder algo-rithmThe values of stability of the candidate genes obtainedfrom theNormFinder analysis are shown inTable 4Themoststable genes were ACTB for goiter and GAPDH for normalthyroid tissue When all samples were analyzed together themost stable genes were SDHA andACTB Stability values andintraintergroup variations are shown respectively in Table 5and Figure 1

4 Discussion

In this study we evaluated 6 reference genes in 14 thyroidspecimens (7 normal thyroid and 7 goiter) using RT-qPCRThe genes with the lowest variations in expression fornormal samples goiter and all samples were respectivelyGAPDH ACTB SDHA and ACTB when evaluated by theNormFinder

According to de Jonge et al [19] a good candidate geneshould present a small coefficient of variation and aMFC lt 2Moreover a mean expression level lower than the maximumexpression level subtracted with 2 SD is a prerequisite for acandidate reference gene By these criteria only B2M andGAPDH genes could be used as internal control for goiterand none of these genes could be used for normal thyroidalthough all had MFC lt 2


Table 3 Dispersion data of raw Cq values for candidate reference genes in goiter and normal thyroid tissue

Tissue Symbol gene Mean Cq SD CV () Minimum Maximum MFC

Goiter

ACTB 2188 178 81 1978 2500 126B2M 2083 259 93 1810 2611 144

GAPDH 2118 195 92 1954 2560 131HPRT1 3010 310 103 2548 3524 138SDHA 3117 221 71 2951 3456 117YWHAY 2748 247 90 2271 3093 136

Normal

ACTB 2356 192 81 1999 2569 129B2M 2463 508 206 1853 3265 176


SD standard deviation CV coefficient of variation and MFC maximum fold change (the ratio of the maximum and minimum values)

Table 4 Stability values of reference genes candidates calculated bythe NormFinder software for each tissue

Tissue Symbol gene Stability value

Goiter

ACTB 0228SDHA 0298HPRT1 0567GAPDH 0685YWHAZ 0688B2M 1711

Normal

GAPDH 0114SDHA 0114ACTB 0221HPRT1 0643YWHAZ 1026B2M 2867

The NormFinder algorithm was used to rank the candi-date reference genes based on their stability values The geneexpression is more stable when this value is closer to zeroThe stability value less than 015 is the cutoff for an acceptablereference gene [4] Considering these criteria none of theselected genes could be used to normalize RT-qPCR datafor goiter tissues and GAPDH and SDHA could be usedfor normal thyroid When all data were analyzed togetherSDHA and ACTB genes had the lowest stability valuesrespectively 0076 and 0082 We suggest using ACTB asreference gene when comparing gene expression in normalthyroid and goiter because this gene had the lowest intra-and intergroup variations Our results differ somewhat fromthose of Chantawibul et al [10] who found the higheststability value for HPRT1 while the GAPDH had the loweststability value These differences may partly be explainedby the sample selection which included goiters adenomascarcinomas and lymphocytic thyroiditis

ACTB mRNA is expressed at moderately abundant levelsin most cell types and its protein plays a key role in the

Table 5 Stability values of reference candidate genes calculated bythe NormFinder software for all tissues

Symbol gene Stability valueSDHA 0076ACTB 0082GAPDH 0150HPRT1 0221YWHAZ 0311B2M 0830

cytoskeleton maintenance Recently some studies have sug-gested that this gene does not satisfy the basic requirementsfor application as internal control due to change in expressionunder various biomedical stimuli [20 21] Neverthelessin normal thyroid cells ACTB expression appeared to bestable in response to different experimental treatments [9]Other studies have demonstrated that ACTB can be used asreference gene [22ndash24] for different cell types

One limitation of this study is the small number ofsamples of goiter nevertheless all samples were evaluated bytwo pathologists who used the same criteria to define goiter

In conclusion the results of the present study suggestthat ACTB gene is more stable than SDHA GAPDH HPRTIYWHAZ and B2M when evaluating human normal thyroidand goiter together

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge FIPEHCPA CAPESPROF and CNPq for financial support


References

[1] M W Pfaffl A Tichopad C Prgomet and T P NeuviansldquoDetermination of stable housekeeping genes differentially reg-ulated target genes and sample integrity BestKeepermdashExcel-based tool using pair-wise correlationsrdquo Biotechnology Lettersvol 26 no 6 pp 509ndash515 2004

[2] S A Bustin ldquoQuantification of mRNA using real-time reversetranscription PCR (RT-PCR) trends and problemsrdquo Journal ofMolecular Endocrinology vol 29 no 1 pp 23ndash39 2002

[3] S A Bustin V Benes J A Garson et al ldquoTheMIQE guidelinesminimum information for publication of quantitative real-timePCRexperimentsrdquoClinical Chemistry vol 55 no 4 pp 611ndash6222009

[4] J Vandesompele K De Preter F Pattyn et al ldquoAccurate nor-malization of real-time quantitative RT-PCR data by geometricaveraging of multiple internal control genesrdquo Genome Biologyvol 3 no 7 Article ID RESEARCH0034 2002

[5] O Thellin W Zorzi B Lakaye et al ldquoHousekeeping genes asinternal standards use and limitsrdquo Journal of Biotechnology vol75 no 2-3 pp 291ndash295 1999

[6] A Radonic SThulke IMMackayO LandtW Siegert andANitsche ldquoGuideline to reference gene selection for quantitativereal-time PCRrdquo Biochemical and Biophysical Research Commu-nications vol 313 no 4 pp 856ndash862 2004

[7] F Haller B Kulle S Schwager et al ldquoEquivalence test inquantitative reverse transcription polymerase chain reactionconfirmation of reference genes suitable for normalizationrdquoAnalytical Biochemistry vol 335 no 1 pp 1ndash9 2004

[8] J Dundas andM Ling ldquoReference genes for measuring mRNAexpressionrdquo Theory in Biosciences vol 131 no 4 pp 215ndash2232012

[9] A P Santin A F Souza L S Brum and T W FurlanettoldquoValidation of reference genes for normalizing gene expressionin real-time quantitative reverse transcription PCR in humanthyroid cells in primary culture treated with progesterone andestradiolrdquo Molecular Biotechnology vol 54 no 2 pp 278ndash2822013

[10] S Chantawibul A Anuwong and K Leelawat ldquoValidationof appropriate reference genes for gene expression studies inhuman thyroid gland using real-time RT-PCRrdquo Journal of theMedical Association ofThailand vol 95 supplement 3 pp S36ndashS40 2012

[11] C Lecchi F Dilda P Sartorelli and F Ceciliani ldquoWidespreadexpression of SAA and Hp RNA in bovine tissues after evalu-ation of suitable reference genesrdquo Veterinary Immunology andImmunopathology vol 145 no 1-2 pp 556ndash562 2012

[12] Q Li K J Domig T Ettle W Windisch C Mair and KSchedle ldquoEvaluation of potential reference genes for relativequantification by RT-qPCR in different porcine tissues derivedfrom feeding studiesrdquo International Journal of Molecular Sci-ences vol 12 no 3 pp 1727ndash1734 2011

[13] P Lisowski M Pierzchała J Goscik C S Pareek and LZwierzchowski ldquoEvaluation of reference genes for studies ofgene expression in the bovine liver kidney pituitary andthyroidrdquo Journal of Applied Genetics vol 49 no 4 pp 367ndash3722008

[14] H Studer H J Peter and H Gerber ldquoNatural heterogeneity ofthyroid cells the basis for understanding thyroid function andnodular goiter growthrdquo Endocrine Reviews vol 10 no 2 pp125ndash135 1989

[15] K Krohn D Fuhrer Y Bayer et al ldquoMolecular pathogenesisof euthyroid and toxic multinodular goiterrdquo Endocrine Reviewsvol 26 no 4 pp 504ndash524 2005

[16] Z W Baloch and V A LiVolsi ldquoFollicular-patterned lesions ofthe thyroid the bane of the pathologistrdquo American Journal ofClinical Pathology vol 117 no 1 pp 143ndash150 2002

[17] A F Souza I S Brum B S Neto M Berger and G BranchinildquoReference gene for primary culture of prostate cancer cellsrdquoMolecular Biology Reports vol 40 no 4 pp 2955ndash2962 2012

[18] C L Andersen J L Jensen and T F Orntoft ldquoNormalization ofreal-time quantitative reverse transcription-PCR data a model-based variance estimation approach to identify genes suited fornormalization applied to bladder and colon cancer data setsrdquoCancer Research vol 64 no 15 pp 5245ndash5250 2004

[19] H J de Jonge R S N Fehrmann E S J M de Bont et alldquoEvidence based selection of housekeeping genesrdquo PLoS ONEvol 2 no 9 article e898 2007

[20] S Selvey EWThompson K Matthaei R A Lea M G Irvingand L R Griffiths ldquo120573-actinmdashan unsuitable internal control forRT-PCRrdquoMolecular and Cellular Probes vol 15 no 5 pp 307ndash311 2001

[21] W Ruan and M Lai ldquoActin a reliable marker of internalcontrolrdquo Clinica Chimica Acta vol 385 no 1-2 pp 1ndash5 2007

[22] R Mori Q Wang K D Danenberg J K Pinski and P VDanenberg ldquoBoth 120573-actin and GAPDH are useful referencegenes for normalization of quantitative RT-PCR in humanFFPEtissue samples of prostate cancerrdquo Prostate vol 68 no 14 pp1555ndash1560 2008

[23] R Mehta A Birerdinc N Hossain et al ldquoValidation ofendogenous reference genes for qRT-PCR analysis of humanvisceral adipose samplesrdquoBMCMolecular Biology vol 11 article39 2010

[24] R Hayashi S Hayashi K Arai M Chikuda and Y ObaraldquoEffects of antioxidant supplementation on mRNA expressionof glucose-6-phosphate dehydrogenase 120573-actin and 18S rRNAin the anterior capsule of the lens in cataract patientsrdquo Experi-mental Eye Research vol 96 no 1 pp 48ndash54 2012

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Stability of genes candidates to normalize RT-qPCR for thyroid derived biological material

Study Material Reference gene stability MethodSantin et al 2013 [9] Human normal cells ACTBgtTBPgtGAPDHgtB2M NormFinderChantawibul et al 2012 [10] Human tissueslowast GAPDHgtACTBgtB2MgtRPLIAgtHPRT1 NormFinder

Lecchi et al 2012 [11] Bovine tissues including thyroid SF3A1=HBMSgtACTB geNormHMBSgtACTBgtGAPDH NormFinder

Li et al 2011 [12] Porcine thyroid tissue 18S rRNAgtUbiquitingtHistone H3gtACTB geNorm18S rRNAgtUbiquitingtHistone H3gtACTBgtGAPDH NormFinder

Lisowski et al 2008 [13] Bovine thyroid tissue TBP=HPRT1gtYWHAZgtACTB geNormlowast13 goiters 6 adenomas 4 carcinomas (2 follicular + 2 papillary) and 2 lymphocytic thyroiditis

follicular cells some nodules have a polyclonal origin andothers a monoclonal origin These changes may be related tomutations in oncogenes which do not produce malignancyper se but would alter growth and function [14 15] Thesealterations could modify the expression of many thyroidgenes including the ones usually described as referencegenes so the evaluation of candidates to normalize RT-qPCRis necessary

Little is known about the expression stability of referencegenes in thyroid cells or tissue as shown in Table 1

Considering the species there are many differences inreference genes stability evaluated in these studies Althoughin human tissues the same software was used to analyzedata differences in RT-qPCR and cells status could havecontributed to these findings In addition a stable expressionof a reference gene in one tumor type does not predict a stableexpression in another tumor type [7] In this way there is nouniversal reference gene being necessary to validate potentialreference genes for any experimental condition So this studyaimed to evaluate themost stable reference gene for RT-qPCRin human normal thyroid and goiter tissues

2 Material and Methods

21 Tissue Acquisition Normal thyroid and goiter tissueswere obtained from patients who underwent total thyroidec-tomy as part of treatment for differentiated thyroid cancerin the Hospital de Clınicas de Porto Alegre (HCPA) Afterthe evaluation of macroscopic and frozen sections of surgicalspecimens by two pathologists samples of normal thyroidor goiter tissue were frozen in liquid nitrogen and stored atminus80∘C until further processing The pattern used to confirmthe presence of goiter was a well-defined fibrous capsulewith a mixture of macrofollicles and microfollicles and insome cases some degenerative changes such as fibrosis andhemorrhage [16] This study was approved by the EthicsCommittee of HCPA (GPPG 12-0272)

22 Nucleic Acid Extraction and Reverse Transcription Nor-mal thyroid and goiter tissue were homogenized mechan-ically with Omnimix for 30 seconds in Trizol (InvitrogenLife Technologies Carlsbad CA USA) and total RNAwas extracted with the same commercial kit according tothe manufacturerrsquos protocol The RNA concentration and

purity were assessed with the NanoDrop 1000 Spectropho-tometer (Thermo Fisher Scientific Wilmington DE USA)Samples were then stored at minus80∘C 1 120583g total RNA wasreverse-transcribed to produce cDNA using random andoligo-dT primers and Superscript II reverse transcriptase(Invitrogen Life Technologies) in total reaction volumes of20120583L All cDNA samples were diluted 10-fold with diethylpyrocarbonate- (DEPC-) treated water and stored at minus20∘C

23 Gene Selection and Quantitative PCR Six genes com-monly used as reference in RT-qPCR gene expression exper-iments were selected (Table 1) beta-actin (ACTB related tocell structure) glyceraldehyde-3-phosphate dehydrogenase(GAPDH related to carbohydrate metabolism) succinatedehydrogenase subunit A flavoprotein (Fp) (SDHA relatedto energy metabolism) hypoxanthine phosphoribosyltrans-ferase I (HPRTI related to nucleotide metabolism) tyrosine3-monooxygenasetryptophan 5-monooxygenase activationprotein zeta polypeptide (YWHAZ related to cell growthand death) and beta-2-microglobulin (B2M related tomajorhistocompatibility complex) Primers sequence shown inTable 2 was described previously [17] Quantification ofamplified samples was performed based on a standard curvewith five successive tenfold dilution points of a pool of cDNAsamples

RT-qPCR was performed using Applied BiosystemsStepOne Real-Time PCR System using Kit Platinum SYBRGreen qPCR SuperMix-UDG (Invitrogen Life TechnologiesCarlsbad CA USA) The RT-qPCR was performed with aninitial denaturation (10min at 95∘C) followed by 40 cyclesof denaturation (15 s at 95∘C) annealing and extension (45 sat 60∘C) Melting curve was acquired to ensure that a singleproduct was amplified in each reaction

24 Statistics To evaluate the stability of the candidatereference genes in thyroid normal and goiter tissues theNormFinder algorithm was used [18] In addition the analy-sis of raw quantification cycle (Cq) values of each gene wasused to evaluate their stability Mean Cq values standarddeviation (SD) coefficient of variation (CV) and maximumfold change (MFC the ratio of the maximum and minimumvalues observed within the dataset) were calculated





sequence












Intr

agro

up v

aria

tion

GoiterNormal

GoiterNormal

Inte

rgro

up v

aria

tion

0150

0100

0050

0000

minus0050

minus0100

minus0150

900

800

700

600

500

400

300

200

100

000


3 Results


4 Discussion






Goiter

ACTB 2188 178 81 1978 2500 126B2M 2083 259 93 1810 2611 144


Normal

ACTB 2356 192 81 1999 2569 129B2M 2463 508 206 1853 3265 176





Goiter


Normal











Acknowledgments



References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















sequence












Intr

agro

up v

aria

tion

GoiterNormal

GoiterNormal

Inte

rgro

up v

aria

tion

0150

0100

0050

0000

minus0050

minus0100

minus0150

900

800

700

600

500

400

300

200

100

000


3 Results


4 Discussion






Goiter

ACTB 2188 178 81 1978 2500 126B2M 2083 259 93 1810 2611 144


Normal

ACTB 2356 192 81 1999 2569 129B2M 2463 508 206 1853 3265 176





Goiter


Normal











Acknowledgments



References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Goiter

ACTB 2188 178 81 1978 2500 126B2M 2083 259 93 1810 2611 144


Normal

ACTB 2356 192 81 1999 2569 129B2M 2463 508 206 1853 3265 176





Goiter


Normal











Acknowledgments



References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Validation of Reference Genes for ...downloads.hindawi.com/journals/bmri/2014/198582.pdfBioMed Research International T : Stability of genes candidates to normalize