Lecture 4-5 (RNA isolation & gene expression analysis) 2024 PDF

1 Gene Expression Analysis  2 Overview of RT-qPCR 1. In RT-qPCR, total RNA (messanger RNA, transfer RNA or ribosomal RNA) or small RNA (ncRNA, miRNA, etc) is first 1. RNA isolation isola...

1 Gene Expression Analysis  2 Overview of RT-qPCR 1. In RT-qPCR, total RNA (messanger RNA, transfer RNA or ribosomal RNA) or small RNA (ncRNA, miRNA, etc) is first 1. RNA isolation isolated. RT 2. RNA is transcribed into complementary DNA (cDNA) by 2. cDNA synthesis reverse transcriptase (RT). fluorescent probe Taq DNA Pol 3. The cDNA is then used as the template in the PCR reaction, together with gene specific primers and a fluorescent probe 3. PCR amplification (such as SYBR Green I, which binds only to double stranded DNA) 4. cDNA is amplified and the fluorescence signal that is 4. Fluorescence generated reflects the amount of PCR product formed (qPCR). quantification  3 Overview of RT-qPCR 1. RNA isolation 5’ AAAAA 5’ AAAAA 5’ AAAAA 2. cDNA synthesis Oligo (dT) Reverse Gene specific 5’ 5’ AAAAA 5’ primers transcriptase (RT) primers 5’ 5’ AAAAA 5’ AAAAA 3. PCR amplification Taq DNA polymerase Gene specific primers  4 1. RNA isolation  5 Precautions to take when working with RNA The ability to isolate clean, intact RNA from cells is essential for experiments that measure transcript levels, for cloning of intact cDNAs, and for functional analysis of RNA metabolism. Working with RNA is more demanding than working with DNA, because of the chemical instability of the RNA and the ubiquitous presence of Ribonucleases (RNases), endoribonucleases that specifically hydrolyze RNA. Unlike DNases, RNases are very stable, do not need metal ion co-factors and can maintain activity even after prolonged boiling or autoclaving.  6 Precautions to take when working with RNA Extract RNA as quickly as possible. Use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at –70°C. Do not let the frozen tissue thaw during handling. This procedure minimizes degradation of RNA, by limiting the activity of endogenous RNases. Always wear gloves. Hands are a major source of contaminating RNase. Clean benches with 100% ethanol. Use sterile, disposable plastic ware, or commercially available RNase-free plastic ware. Glassware should be baked at +180 to 300°C for at least 4 hours. Autoclaving glassware is not sufficient to eliminate RNases. ALL solutions should be made with DEPC-treated H2O. Diethyl pyrocarbonate (DEPC) inactivates RNase. If RNA will be transferred to a membrane (Northern blot) electrophoresis tanks for RNA analysis can be cleaned with 1% SDS, rinsed with H 2O, rinsed with absolute ethanol, and finally soaked in 3% H2O2 for 10 minutes. Rinse tanks with DEPC-treated H2O before use. (From: Roche).  7 RNA isolation steps The first step in all RNA isolation protocols therefore involves lysing the cell in a chemical environment that results in denaturation of RNases. Most protocols use mild detergents, or phenol + SDS, or guanidinium isothiocyanate. The latter is method of choice for RNA isolation from plants. The RNA is then fractionated from the other cellular macromolecules under conditions that limit or eliminate any residual RNase activity. Several kits for RNA isolation are commercially available. These kits are easy to use and work well. However, they are usually more expensive.  8 RNA isolation with Qiagen RNeasy kit Biological samples are first lysed and homogenized in the presence of a denaturing buffer containing guanidine-thiocyanate and Beta-mercaptoethanol (ß-ME), both of which immediately inactivate (denature) RNases to ensure purification of intact RNA. Ethanol is added to provide appropriate binding conditions. The high-salt buffer allows RNA longer than 200 bases to bind to a silica membrane. Provides an enrichment for mRNA, since most RNAs