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

Author(s): Liedtke W, Battistini L, Brosnan CF, Raine CS

Publication: PCR Methods Appl, 1994, Vol. 4, Page 185-7

PubMed ID: 7580904 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess the impact of peripheral blood mononuclear cell cryopreservation, and cell washing and centrifugation prior to lysis on resultant data from reverse transcription polymerase chain reaction (RT-PCR) analysis. Several analytical parameters were also examined including total RNA versus poly(A) RNA as template, temperature of the RT reaction, RT priming method, and RNA extraction methods.The genes amplified were V delta 2-J delta 1, beta-actin, and CD3.

   Summary of Findings:

   The authors report that centrifugation and pelleting of cells enabled lower template dilutions than direct exposure to the lysis agent; washing pellets in Hank's balanced saline solution yielded no advantage over unwashed pellets, and frozen cells performed as well as fresh preparations. The authors also identified the following analytical parameters to be beneficial in RT-PCR analysis of RNA extracted from PBMC: commercially available RNA extraction kits (Iowa Biotech Corp.; MRC, Inc.; Biotecx, Inc.; Invitrogen Corp.) yielded superior results to published protocols, oligo(dT) and RT-PCR downstream primers were preferable to random hexamer primers, and a RT temperature of 43 degrees C increased yield compared to 37 degrees C. Use of poly(A) RNA and total RNA yielded similar results as did PCR master mixes containing potassium glutamate or potassium chloride.

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell capture method	Centrifugation (cell pellet) No centrifugation
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Snap frozen
   Analyte Extraction and Purification	Analyte isolation method	Poly(A) RNA Total RNA
   RT-PCR Specific	Priming method	Random hexamer Oligo (dT) RT-PCR downstream priming
   RT-PCR Specific	RNA incubation temperature	37 degrees C 43 degrees C
   RT-PCR Specific	Reaction solution	Potassium glutamate Potassium chloride

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