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Validation of an appropriate reference gene for normalization of reverse transcription-quantitative polymerase chain reaction data from rectal cancer biopsies.: Normalization of RT-qPCR data in rectal cancer

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HAL Id: inserm-00396213

https://www.hal.inserm.fr/inserm-00396213

Submitted on 19 Jun 2009

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Validation of an appropriate reference gene for

normalization of reverse transcription-quantitative

polymerase chain reaction data from rectal cancer

biopsies.

Alexandre Ho-Pun-Cheung, Caroline Bascoul-Mollevi, Eric Assénat, Frédéric

Bibeau, Florence Boissière-Michot, Dominic Cellier, Marc Ychou, Evelyne

Lopez-Crapez

To cite this version:

Alexandre Ho-Pun-Cheung, Caroline Bascoul-Mollevi, Eric Assénat, Frédéric Bibeau, Florence Boissière-Michot, et al.. Validation of an appropriate reference gene for normalization of reverse transcription-quantitative polymerase chain reaction data from rectal cancer biopsies.: Normalization of RT-qPCR data in rectal cancer. Analytical Biochemistry, Elsevier Masson, 2009, 388 (2), pp.348-50. �10.1016/j.ab.2009.03.001�. �inserm-00396213�

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Supplementary Material

Supplementary Table 1

Genes examined

Gene

symbol

Gene name

GenBank

accession

no.

Function

Relative

expression

level

Control gene (assessment of RT-qPCR inhibitors)

CAB A. thalianabinding protein chlorophyll a/b- X56062 – – Candidate reference genes

18S 18S ribosomal RNA X03205 Part of a ribosomal subunit Very high

28S 28S ribosomal RNA M11167 Part of a ribosomal subunit Very high

ACTB Beta-actin NM_001101 Cytoskeletal protein High ATP6 ATP synthase F0 subunit 6 NC_001807.4 Subunit of mitochondrial ATP synthase High GAPDH Glyceraldehyde-3-phosphate dehydrogenase NM_002046 Glycolysis enzyme High ATUB Alpha tubulin NM_006082 Component of microtubules Medium

B2M Beta-2-microglobulin NM_004048 Beta chain of major histocompatibility complex antigen Medium

PGK1 Phosphoglycerate kinase 1 NM_000291 Glycolysis enzyme Medium RPLP0 Ribosomal protein, large, P0 NM_001002 Component of the 60S subunit Medium POLR2L Polymerase RNA II polypeptide L NM_021128 Subunit of RNA polymerase II Low PSMB6 Proteasome subunit Y NM_002798 Proteasome subunit Low HMBS Hydroxymethylbilane synthase NM_001024382 Enzyme involved in heme synthesis Very low HPRT Hypoxanthine phosphoribosyl transferase 1 NM_000194 Central role in the generation of purine nucleotides Very low PPIE Peptidylprolyl isomerase E NM_203457 Involved in the folding of proteins Very low

TBP TATA box binding protein NM_003194 Initiation of transcription by RNA polymerase II Very low

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Supplementary Table 2

Characteristics of the real-time PCR assays

Gene

Primer sequences (5’



3’)

spanning

Intron-

Amplicon

size (bp)

Temperature

Annealing

(°C)

qPCR

efficiency

(%)

Control gene (assessment of RT-qPCR inhibitors)

CAB F : CCATTGCATTTGTTGAGCAC R : CAATTCCTCGAGCTTCTTGG Useless 119 62 100%

Candidate reference genes

18S F : GGCGCCCCCTCGATGCTCTTAG R : GCTCGGGCCTGCTTTGAACACTCT No 89 60 98%

28S F : GGGGGAGAGGGTGTAAATCT R : CAGAGGCTGTTCACCTTGG No 73 62 95%

ACTB F : CTGTGGCATCCACGAAACTA R : AGTACTTGCGCTCAGGAGGA Yes 200 60 100% ATP6 F : AATGCCCTAGCCCACTTCTT R : CCAGGGCTATTGGTTGAATG No 103 62 100% GAPDH F : TGCACCACCAACTGCTTAGC R : GGCATGGACTGTGGTCATGAG Yes 87 64 100% ATUB F : TTACCTCGACTCTTAGCTTGTCG R : GGATGGAGATGCACTCACG Yes 107 60 92%

B2M F : CACCCCCACTGAAAAAGATG R : ATATTAAAAAGCAAGCAAGCAGAA Yes 167 60 93%

PGK1 F : GGGCTGCATCACCATCATA R : CCCCAGGAAGGACTTTACCTT Yes 137 62 100% RPLP0 F : CACTGAGATCAGGGACATGTTG R : CTTCACATGGGGCAATGG Yes 113 62 100% POLR2L F : CAACAAGTGGGAGGCTTACCT R : AGCTTCTCGATCAGGTCCAC Yes 132 64 98% PSMB6 F : GATACCGGGAAGACCTGATG R : AATGGCAAAGGACTGCCTTA Yes 116 60 99% HMBS F : TGTGGTGGGAACCAGCTC R : TGTTGAGGTTTCCCCGAAT Yes 92 64 100% HPRT F : TGATAGATCCATTCCTATGACTGTAGA R : AAGACATTCTTTCCAGTTAAAGTTGAG Yes 126 62 94% PPIE F : GCGTTCATTCCTTTTGGAGA R : AAATCCTCGGTGCTTTTCTG Yes 75 60 99%

TBP F : AAAGACCATTGCACTTCGTG R : GCACACCATTTTCCCAGAAC Yes 124 52 100%

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