A two-step method for the extraction of high-quality RNA from endoscopic biopsies*
U. Lendeckel, P. Malfertheiner, Th. Wex, G. Treiber,
Background: The usage of molecular techniques such as
quantitative RT-PCR depend on the quality of cellular RNA.
In particular, RNA extraction from endoscopic biopsies is
difficult with respect to yield and, especially, integrity.
Therefore, we developed a method that allows extraction of
high-quality RNA from these sources.
Methods: Endoscopic biopsies taken from the gastric antrum,
corpus and duodenum were subjected to various RNA
extraction protocols, and the RNA was used for quantitative
Results: The subsequent usage of two methods, (i) a phenol/
chloroform extraction and (ii) a colunn-based extraction
method resulted in a yield of 4.5ìg total RNA/biopsy
of reliable quality in 80% of samples. The quantitative RTPCR
analysis revealed that only RNA samples that clearly
show both 18S- and 28S-RNA bands in agarose gel electrophoresis
were suitable for quantitative RT-PCR. In all these
samples, both the corpus-specific pepsinogen C-mRNA and
the duodenum-specific mdr-1-mRNA could be consistently
detected. In degraded RNA, pepsinogen C, mdr-1 or â-actin
mRNAs were still detectable, but the quantitative
det4rmination gave inconsistent data. Conclusions: The two-step method described here proved
to be the most reproducible approach to obtaining sufficient
amounts of high-quality RNA from endoscopic biopsies
as required for quantitative gene expression analyses.