Extraction of RNA/cDNA and Genomic DNA from Tissue with Real-Time PCR

With kind permission of Christian M. Leutenegger, Ph.D., University of California, Davis.

Sample Collection, Preparation and Tissue Grinding 

Fresh samples of animal tissue were collected, trimmed to approximately 50 – 100 mg of wet weight, snap-frozen in liquid nitrogen, and stored at –80oC. The animals were human, dog, cat, mouse, cow, and horse, as well as fish and clams; see Table 1. Before DNA and/or RNA extraction, the tissues were transferred frozen to a deep-well titer plate standing on a block of dry ice. Each well contained two 4-mm stainless steel balls (SPEX SamplePrep cat. no. 2150) and 500 microliters of buffer (Applied Biosystems nucleic acid purification lysis buffer). The plates were sealed with a plastic cover and subjected to grinding in the Geno/Grinder for 2 minutes at a setting of 1000 strokes per minute. After 30 minutes at 4oC, lysates were used for either gDNA extraction or total RNA extraction. Conditions were optimized for an Applied Biosystems 6700 automated nucleic acid (ANA) workstation, according to the manufacturer’s instructions. The final amount of tissue subjected to RNA and/or DNA extractions was between 10 and 20 mg.

Quality Control of Extracted RNA/cDNA and Genomic DNA Using Real-Time Taqman® PCR 

To assess the quality of the extracted RNA, complementary DNA (cDNA) was synthesized using  Invitrogen products: 200 units of SuperScript III, 600 ng of random hexadeoxyribonucleotide  (pd(N)6) primer (random hexameter priner), 10 U RnaseOut (Rnase inhibitor), and 1 mM dNTPs in a final volume of 40 µL. The reverse transcription reaction took place for 50 minutes at 50ºC. After addition of 60 µL of water, the reaction was terminated by heating for 5 minutes to 95ºC and cooling on ice. The quality of the cDNA is judged according to the CT value obtained with a endogenous control TaqMan PCR system from a defined amount of tissue. Samples with values above a certain threshold indicate impaired sample quality and degradation of total RNA and warrant re-extraction of a back-up sample. To assess the quality of cDNA, we normally use TaqMan PCR systems targeting species specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal genes (such as 18S rRNA or ITS-2). 

To assess the quality of extracted genomic DNA, species specific TaqMan PCR systems were developed targeting single copy genes to allow the quantitation of genome equivalents and cell numbers. An overview of TaqMan systems used for gDNA quality control is given in Table 1. The gDNA quality from a defined amount of tissue is judged according to the CT value using a TaqMan PCR system targeting a single copy gene. Samples with values above a certain threshold indicate degraded DNA and warrant re-extraction of a back-up sample. GAPDH TaqMan systems can be used to target the single copy GAPDH pseudogene (Galland et al., 1990; Garcia-Meunier et al., 1993). 

Each real-time TaqMan PCR reaction contained 400 nM of each primer, 80 nM of the TaqMan probe and commercially available PCR mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems) containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5 mM deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction and 5 µL of the diluted cDNA sample or the gDNA in a final volume of 25 µL. The samples were placed in 96 well plates and amplified in an automated fluorometer (ABI PRISM 7700 Sequence Detection System, Applied Biosystems). Amplification conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C.

References 

GAPDH pseudogenes: 

Galland, F., M. Stefanova, V. Pirisi, and D. Birnbaum. 1990. Characterization of a murine glyceraldehyde-3-phosphate dehydrogenase pseudogene. Biochimie. 72:759-62. 

Garcia-Meunier, P., M. Etienne-Julan, P. Fort, M. Piechaczyk, and F. Bonhomme. 1993. Concerted evolution in the GAPDH family of retrotransposed pseudogenes. Mamm. Genome. 4:695-703. Leutenegger, C.M. , B. von Rechenberg, K. Zlinsky, C. Mislin, M. Akens, J. Auer, and H. Lutz: 

Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate. Calcified Tissue International, 65:378-383, 1999. 

Leutenegger, C.M., A.M. Alluwaimi, W. Smith, L. Perani, J.M. Cullor. Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. Veterinary Immunology and Immunopathology. 77: 275-287, 2001. 

Leutenegger, C.M.. The real-time TaqMan PCR and applications in Veterinary Medicine. Veterinary Sciences Tomorrow, Online Journal, Jan 1, 2001.