A two-step method for the extraction of high-quality RNA from endoscopic biopsies.
The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR 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 as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.
Wex
Thomas
T
Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany. thomas.wex@medizin.uni-magdeburg.de
Treiber
Gerhard
G
Lendeckel
Uwe
U
Malfertheiner
Peter
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000199:Actins / Q000235:genetics; D001706:Biopsy; D015152:Blotting, Northern; D004386:Duodenum / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D016099:Endoscopy, Gastrointestinal; D005753:Gastric Mucosa / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008722:Methods; D020115:Pepsinogen C / Q000235:genetics; D011706:Pyloric Antrum / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D020115:Pepsinogen C; D012313:RNA