A comparison of methods for RNA extraction from lymphocytes for RT-PCR.

1Albert Einstein College of Medicine, Department of Pathology, Division of Neuropathology, Bronx, New York 10461; 2University of Essen, Department of Neurology, 45122 Essen, Germany; 3University "La Sapienza", Department of Neurology, Rome, Italy Various methods have been described to extract RNA from nonadherent mammalian cells or are provided in protocols accompanying various commercial ly available reagents. (1-1~ Reverse transcriptase-PCR (RT-PCR) allows the amplification, even the quantitation, of previously undetectable amounts of m R N A . (11'1z) In this burgeoning field, some major points of interest have not yet been investigated: (1) comparison of the cited methods (given the importance of RT-PCR, l imit ing dilutional assays of cDNA transcripts is a better end point than other RNA parameters like spectrophotometry, Northern blotting, etc.); (2) whether total RNA or poly(A) RNA extraction is more favorable for subsequent RT-PCR; and (3) a detailed comparison of cDNA synthesis with oligo(dT), random hexamer, or RT-PCR downstream primers. We have addressed these questions by extracting RNA from h u m a n lymphocytes. The extracted RNA [either poly(A) or total] was reverse-transcribed, and the cDNA was subjected to PCR-amplifying [3-actin, CD3, and ~//BTCR rearrangement VB2J~l.(~3-15~ MATERIALS AND METHODS Lymphocytes