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中生网 > 实验技术 > PCR技术 > Nested RT-PCR for Hepatitis C from Paraffin Sections

Nested RT-PCR for Hepatitis C from Paraffin Sections

更新:2013年10月10日 阅读次数: 【字体:

RNA Extraction from Histologic Sections
Unstained 4 µm thick sections of formalin fixed paraffin embedded liverbiopsies were transferred from glass slides to 1.5 ml non-siliconizedmicrocentrifuge tubes containing 200µl of TRIzol (Gibco BRL,Gaithersburg MD), tapped thoroughly to immerse the tissue section andwarmed to 65°C for ten minutes, then placed on ice for one minute. Tenµg of E. coli tRNA and 20µl of chloroform were added, and after mixingthe tubes were incubated for 3 minutes at room temperature. The sampleswere spun in a countertop centrifuge at 4°C for 15 minutes, and 100µl ofthe clear aqueous upper phase was transferred to a new microcentrifugetube. After addition of 100µl of isoproponal the tubes were incubatedfor 10 minutes at room temperature, then centrifuged 12,000 rpm at 4°Cfor 10 minutes. The supernatant was carefully decanted and the RNApellet was washed with 100µl of 75% ethanol. The pellets were driedunder vacuum (without centrifugation or heat) for 5 minutes to less thandryness, then resuspended in 50µl of ddH2O.
Oligonucleotide Primers
"Multiplex" amplification was performed for the 5''non-coding region of the hepatitis C RNA genome[1] as well as albuminmRNA to demonstrate successful extraction of RNA and subsequent RT-PCRusing primers to span two introns.[2]
First Round Primers:
Hepatitis C (140 bp)
GGCGACACTCCACCATAGATCA (upstream) GGTTCCGCAGACCACTATGGC (downstream)Albumin mRNA (206 bp)
GCTGCTTTTACAGAATGTTGCCAA (upstream) ACTTCTGCAAACTCAGCTTTGGG (downstream)Second Round Primers:
Hepatitis C (82 bp)
CTCCCCTGTGAGGAACTAC (upstream) GGTCCTGGAGGCTGCACA (downstream) Albumin mRNA (151 bp)
ATAAAGCTGCCTGCCTGTTG (upstream) AATCTCTGGCTCAGGCGAG (downstream) PCR Reaction and Cycling Conditions:
RT-PCR is performed with 23 µl of the extracted material from eachslide in a final 50 µl reaction mix containing 200 µM of each dNTP, 2.5ng/µl (approximately 380 nM) of each first round primer, 2.5u of Taqpolymerase, 1X Taq polymerase buffer, 4 mM MgCl2, 0.5µl (12.5u) of RTAMV (12.5u) and 1µl (40u) RNase inhibitor (both from BoehringerMannheim, Indianapolis IN) using an initial 30 minute reversetranscriptase incubation at 42°C and 3 minute denaturation step followedby 35 cycles of PCR consisting of one minute at 94°C (denaturation), twominutes at 62°C (annealing), and two minutes at 72°C (extension) with afinal 10 minute completion step at 72°C.
Ten percent (5 µl) of the PCR product is then transferred to a secondround reaction mix with second round primers for hepatitis C and albuminmRNA and reagents in the same concentrations as above except for 1.5 mMMgCl2. PCR cycling conditions are as for first round amplificationexcept for an annealing temperature of 54°C. The resulting PCR productcan be resolved by electrophoresis through an agarose gel and directlyvisualized with ethidium bromide staining and ultraviolet illumination.The lengths of the nested PCR products are 82 bp (hepatitis C) and 151bp (albumin).
 
Sensitivity and Specificity:
In a comparative analysis, this RT-PCR protocol was positive in 48 of49 biopsies from patients with circulating anti-hepatitis C antibodies,and no examples of false-positive amplification were seen in 23 negativecontrols liver biopsies. In contrast, 53% of liver biopsies withdocumented hepatitis C infection were negative with immunohistochemistryusing TORDJI-22.
References:
[1.] Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, SugimuraT, Shimotohno K: Molecular cloning of the human hepatitis C virus genomefrom Japanese patients with non-A, non-B hepatitis. Proc. Natl. Acad.Sci USA 87:9524, 1990
[2.]McDonnell MW, Scheiman JJM, Traber PG: Induction of CytochromeP450IA Genes (CYP1A) by Omeprazole in the Human Alimentary Tract.Gastroenterology 103:1509, 1992.
[3.]Svoboda-Newman SM, Greenson JK, Singleton TP, Sun R, Frank TS:Detection of hepatitis C in paraffin sections of formalin fixed liverusing RT-PCR. Modern Pathology, 9 (1):137A, 1996.
 

关键词:RT-PCR
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