A standardized approach to PCR-based semiquantitation of multiple cytokine gene transcripts from small cell samples.
A simple, rapid, reproducible, and nonradioisotopic method for semiquantitative analysis of cytokine mRNAs based on polymerase chain reaction (PCR) is described. RNA isolated from 2.5 million cells has proven sufficient to perform semiquantitative analysis of mRNA for 10 different cytokines. By this approach accurate assessment of mRNA levels for multiple cytokines can be made from as little as 2 ml of blood or about 3 mg of biopsy material. Total cellular RNA is quantitatively recovered by guanidinium isothiocyanate-acid-phenol extraction of a constant number of cells. Further quantitation of RNA is unnecessary. Highly reproducible PCR product formation occurs after specific amplification of aliquots of reverse transcribed test RNA. The photographic image of the ethidium bromide-stained gel accurately reflects the amount of PCR product loaded, both densitometrically and visually. PCR product generation is not affected by the presence of carrier RNA. Thus quantitative recovery of total RNA is possible even from a very small number of cells. Similarly, presence of a large excess of nonspecific RNA from nonexpressing cells does not affect amplification of the specific mRNA under study. A linear relationship between mRNA frequency and PCR product formation is observed over a 256- to 512-fold range. The actual mRNA concentration for each cytokine varies depending on the relative abundance of mRNA for that cytokine relative to total RNA. By performing two amplification cycles (28 and 35) on undiluted and 10-fold diluted RNA samples, the range of detection linearity is extended over a 5000-fold difference in input RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Lagoo-Deenadayalan
S
S
Department of Surgery, University of Alabama, Birmingham.
Lagoo
A S
AS
Barber
W H
WH
Hardy
K J
KJ
D001483:Base Sequence; D002452:Cell Count; D016207:Cytokines / Q000032:analysis*; D004996:Ethidium; D005782:Gels; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012685:Sepharose; D014158:Transcription, Genetic
D016207:Cytokines; D005782:Gels; D012333:RNA, Messenger; D012685:Sepharose; D004996:Ethidium