High-copy cDNA amplification of minimal total RNA quantities for gene expression analyses.
This protocol describes a PCR-based cDNA amplification technique of small total RNA quantities, optimized for determination and verification of gene expression variations in cells or tissue specimen. A proportional amplification of rare and abundant transcripts is thereby achieved by initial random hexamer-primed reverse transcription of total RNA. Compared to established oligo(dT)-primed techniques, this approach generates shorter than full length copies of long RNAs which leads to a normalized cDNA pool for a more adequate PCR-amplification. Subsequent double oligo(dA) tailing of the synthesized total cDNA strands and the utilization of heteropolymeric primers allow a highly specific, up to 500-fold PCR-amplification of the total cellular RNA amount. Thus, obstacles in availability of RNA from limited sources, such as human biopsies or microdissected histological sections, can be overcome. The amplified total cDNA (atcDNA) is shown to be applicable for confirmation of differential gene expression, as demonstrated in this protocol by expression analysis of the multidrug resistance-associated genes mdr1, mrp1 and lrp, using human cell lines as well as microdissected human tissue sections.
Schwabe
H
H
Max-Delbrück-Center for Molecular Medicine, Dept. Surgery/Surgical Oncology, Berlin, Germany. hschwabe@mdc-berlin.de
Stein
U
U
Walther
W
W
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA