University:
Minnesota State University Moorhead
Course:
BIOL-350 | Microbiology
Academic year:

2024
Views:

38

Pages:

21
Author:

Kaushik Sen

RNA EXTRACTION & AMPLIFICATION Made By : Kaushik Sen HCV virus EDTA blood for HCV RNA RT PCR Lysis of cell wall or cell membrane Lysis of nuclear membrane Isolation of nucleic acid Precipitation of RNA Steps of RNA extraction According to RNA extraction kit Extracted RNA Washing Elution Sample: EDTA plasma Sample (Plasma) containing HCV gene Sample (200 μL) + Trizol (800 μL) Trizol The addition of chloroform creates a biphasic system in which DNA and protein partition into the lower organic phase while the upper aqueous phase retains the RNA. It is used to promote phase separation so that RNA is isolated from DNA and proteins in a biological sample. Chloroform 200 μL Chloroform is added Centrifuge tube at 12000 RPM for 10 Min After centrifugation 3 layers are formed Transfer Aqueous phase into another tube The role of isopropanol in RNA isolation is to precipitate RNA. RNA is insoluble in alcohols such as isopropanol and ethanol. Silicon membrane filter Spin Column Collection tube RNAs are attached in Membrane WASH BUFFER Membrane Filter Centrifugation Column Transfer Washing 2X with Washing Buffer ELUTION BUFFER Centrifugation Addition of Elution Buffer Elution of RNA is complete Extracted RNA is stored at -70 PCR reagent & its content Steps of HCV RNA amplification extraction According to 1 Reverse transcriptase 2 DNA polymerase 3 Primer 4 Probe 5 dNTP Deoxyribonucleoside triphosphates 6 Primer HCV RT-PCR kit PCR Hood 96 wells Thermal cycler Bio Rad CFX Opus 96 PCR tubes are being loaded Steps of Amplification 1. Reverse Transcription 2.Annealing 3.Elongation cDNA 4. Denaturation of cDNA at 95° C 5.Annealing at 55° C 6.Elongation at 72° C Detection 1 2 3 4 5 1 2 RFU 3 4 5 No of cycles HCV gene curve IC curve Stnd. curve for HCV gene Stnd. curve for IC Calculation for Viral Load • Generally Sample vol is 0.2 ml • Elution Vol is 60 microliter