mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts

doi:10.1016/S0003-2697(02)00058-1

Analytical Biochemistry

Volume 307, Issue 2, 15 August 2002, Pages 304-315

https://doi.org/10.1016/S0003-2697(02)00058-1 Get rights and content

Abstract

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original “RNA” extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence—which lacks introns but contains a poly(A) tail—for commonly studied internal reference genes β-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design “RNA-specific” PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these “housekeeper” genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a unique tag sequence not otherwise existing in the human genome. Genomic DNA does not incorporate this RT-inserted unique tag. PCR is then performed using a transcript-specific forward primer and a reverse primer that is identical to the unique tag incorporated at RT. Only cDNA made with this RT primer is compatible with this reverse PCR primer, thus eliminating confounding signal from contaminating gDNA. This method performs RNA-specific qualitative and quantitative evaluation of gene expression, while preserving the sensitivity of standard RT-PCR techniques. Applications to low-copy transcripts in human samples are demonstrated.

Section snippets

Materials and methods

RNA isolation. Human lung specimens were flash frozen to −150 °C in the operating theatre within 15 min of surgical resection for clinical purposes, under IRB-approved protocol. Total RNA was extracted from approximately 100 mg of frozen lung tissue using a thiocyanate guanidinium-based method (TRI Reagent protocol, Molecular Research Center, Cincinnati, OH). Lung tissue was fractionated and pulverized on liquid nitrogen to minimize RNA degradation. Frozen pulverized lung tissue was immediately

DNase treatment

All attempts to treat RNA with DNase, prior to RT, resulted in either a loss of RNA or incomplete degradation of gDNA, or both (Fig. 1). Several methods were attempted, including one published protocol recommended for use in quantitative RT-PCR [5] and displayed in the early experiment depicted in Fig. 1. In this DNase treatment example, CYP1A1 was chosen as target transcript because there is no known pseudogene, nor have we or others observed experimental evidence to the contrary. Also, these

Discussion

The sensitivity of RT-PCR has made it an essential tool of molecular biology. With the advent of real-time quantitative PCR technology, transcripts can now be quantitated with great precision. However, experimental controls suggest that processed pseudogene sequences in genomic DNA contaminants of “RNA” extracts can confound even the most well-designed of standard PCR primers. These gDNA sequences are particularly prevalent for highly expressed reference housekeeper genes, such as β-actin,

Acknowledgements

We thank Drs. Michael Fasco and Erasmus Schneider for helpful discussions and Dr. Laurence Kaminsky for laboratory support, and we acknowledge the Molecular Genetics Core of the Wadsworth Center for oligo synthesis and sequencing. The study was supported by NIH K08 ES-00298. A provisional patent application on this method is pending.

References (22)

A.J. Mighell et al.
Vertebrate pseudogenes
FEBS Lett.
(2000)
M. Shibutani et al.
Methacarn fixation: a novel tool for analysis of gene expression in paraffin-embedded tissue specimens
Lab. Invest.
(2000)
L. Overbergh et al.
Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR
Cytokine
(1999)
T. Lion
Current recommendations for positive controls in RT-PCR assays
Leukemia
(2001)
T. Raff et al.
Design and testing of β-actin primers that do not co-amplify processed pseudogenes
BioTechniques
(1997)
K.-A. Kreuzer et al.
Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of B-actin transcripts as quantitative reference
Clin. Chem.
(1999)
Getting Rid of Contaminating DNA. Ambion Tech Notes Newslett. 8 (1) (2001). www.Ambion....
Z. Huang et al.
Optimization of DNase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR
BioTechniques
(1996)
P. Bauer et al.
Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase digestion
BioTechniques
(1997)
K. Ivarson et al.
Evaluation of the effects of DNase treatment on signal specificity in RT-PCR and in situ RT-PCR
BioTechniques
(1998)

J. Leavitt et al.

Molecular cloning and characteristics of mutant and wild type B-actin alleles

Mol. Cell. Biol.

(1984)

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mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts

Abstract

Section snippets

Materials and methods

DNase treatment

Discussion

Acknowledgements

FEBS Lett.

Lab. Invest.

Cytokine

Current recommendations for positive controls in RT-PCR assays

Leukemia

Design and testing of β-actin primers that do not co-amplify processed pseudogenes

BioTechniques

Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of B-actin transcripts as quantitative reference

Clin. Chem.

Optimization of DNase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR

BioTechniques

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase digestion

BioTechniques

Evaluation of the effects of DNase treatment on signal specificity in RT-PCR and in situ RT-PCR

BioTechniques

Molecular cloning and characteristics of mutant and wild type B-actin alleles

Mol. Cell. Biol.