Limitations and modifications of quantitative polymerase chain reaction. Application to measurement of multiple mRNAs present in small amounts of sample RNA.
Quantification of mRNA by polymerase chain reaction (PCR) is performed by using a competitor DNA standard in the PCR or by employing an internal standard RNA following simultaneous reverse transcription (RT) with the sample RNA. The latter approach is more reliable since it accounts for variations in both the RT and PCR steps. However, we describe in this report that at times the internal standard RNA competes with the target mRNA in the PCR when both are present in disproportionate concentrations in the initial simultaneous RT reaction. To overcome the competition in the PCR, multiple simultaneous RT reactions with the sample RTA and the internal standard RNA are required. Such procedures make the approach time consuming and restrict the use of internal standard RNA for quantification of multiple mRNAs present in small amounts of sample RNA. These limitations are circumvented by the competitor DNA standard approach and mRNA levels can be quantified by calculating the RT efficiency. We illustrate the situations by quantifying the levels of IL-2, IL-4, IL-5, IFN-gamma, and beta-actin mRNAs in mitogen stimulated murine T cells using a multiple mRNA specific internal standard.
Babu
J S
JS
Department of Microbiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37996-0845.
Kanangat
S
S
Rouse
B T
BT
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger; D012313:RNA