Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability

Experimental and Molecular Pathology

Volumn 87, Issue 2, 2009, Pages 146-151

  Nardon, Ermanno ^a,b   Donada, Marisa ^b   Bonin, Serena ^a,b   Dotti, Isabella ^a   Stanta, Giorgio ^a,b  

^a UNIVERSITY OF TRIESTE   (Italy)

^b INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY   (Italy)

Author keywords

Formalin fixed and paraffin embedded tissues; Random primers; Real time qRT PCR; Reverse transcription

Indexed keywords

COMPLEMENTARY DNA; MESSENGER RNA; RNA;

ARTICLE; AVIAN MYELOBLASTOSIS VIRUS; CLINICAL ARTICLE; COLON CANCER; CONCENTRATION RESPONSE; CONTROLLED STUDY; HISTOPATHOLOGY; HUMAN; HUMAN TISSUE; INTERMETHOD COMPARISON; MOLONEY LEUKEMIA ONCOVIRUS; NUCLEOTIDE SEQUENCE; QUANTITATIVE ANALYSIS; RELIABILITY; REPRODUCIBILITY; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; RNA EXTRACTION; SENSITIVITY AND SPECIFICITY; TISSUE FIXATION; TUMOR BIOPSY;

DNA PRIMERS; DNA, COMPLEMENTARY; GENE EXPRESSION PROFILING; HUMANS; PARAFFIN EMBEDDING; REPRODUCIBILITY OF RESULTS; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; SENSITIVITY AND SPECIFICITY; TISSUE FIXATION;

AVIAN MYELOBLASTOSIS VIRUS; MOLONEY MURINE LEUKEMIA VIRUS;

EID: 70349270564     PISSN: 00144800     EISSN: 10960945     Source Type: Journal    
DOI: 10.1016/j.yexmp.2009.07.005     Document Type: Article

References (19)

1. Bustin S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25 (2000) 169-193
2. Bustin S.A., and Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15 (2004) 155-166
3. Fuchs B., Zhang K., Rock M.G., Bolander M.E., and Sarkar G. High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR. Mol. Biotechnol. 12 (1999) 237-240
4. Godfrey T.E., Kim S.H., Chavira M., Ruff D.W., Warren R.S., Gray J.W., and Jensen R.H. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction. J. Mol. Diagn. 2 (2000) 84-91
5. Kidd E.A., Yu J., Li X., Shannon W.D., Watson M.A., and McLeod H.L. Variance in the expression of 5-Fluorouracil pathway genes in colorectal cancer. Clin. Cancer Res. 11 (2005) 2612-2619
6. Lekanne Deprez R.H., Fijnvandraat A.C., Ruijter J.M., and Moorman A.F. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal. Biochem. 307 (2002) 63-69
7. Lewis F.a.M., and N.J. Extraction of total RNA from formalin-fixed paraffin-embedded tissue. In: Bustin S.A. (Ed). A-Z of Quantitative RCR (2004), International University Line, LaJolla, CA 591-601
8. Nolan T., Hands R.E., and Bustin S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1 (2006) 1559-1582
9. Peccoud J., and Jacob C. Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys. J. 71 (1996) 101-108
10. Pfaffl M.W. Quantification strategies in real-time PCR. In: Bustin S.A. (Ed). A-Z of Quantitative RCR (2004), International Univ. Line, LaJolla, CA 87-112
11. Ririe K.M., Rasmussen R.P., and Wittwer C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245 (1997) 154-160
12. Specht K., Richter T., Muller U., Walch A., Werner M., and Hofler H. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am. J. Pathol. 158 (2001) 419-429
13. Stahlberg A., Hakansson J., Xian X., Semb H., and Kubista M. Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50 (2004) 509-515
14. Stahlberg A., Kubista M., and Pfaffl M. Comparison of reverse transcriptases in gene expression analysis. Clin. Chem. 50 (2004) 1678-1680
15. Stangegaard M., Dufva I.H., and Dufva M. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA. Biotechniques 40 (2006) 649-657
16. Stanta, G., Schneider, C., 1991, RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification. Biotechniques 11, 304, 306, 308.
17. Stanta G., Bonin S., and Utrera R. RNA quantitative analysis from fixed and paraffin-embedded tissues. Methods Mol. Biol. 86 (1998) 113-119
18. Suslov O., and Steindler D.A. PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency. Nucleic Acids Res. 33 (2005) e181
19. Zhang J., and Byrne C.D. Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. Biochem. J. 337 (1999) 231-241