RNA ISOLATION AND cDNA PREPARATION

Principle
Materials Required
Procedure
Phase Separation
RNA Precipitation
RNA Wash
Redissolving RNA
cDNA Preparation
mRNA Extraction
cDNA Construction
Uses of DNA Library

1. IMMUNE AND MOLECULAR DIAGNOSTIC TOPIC:RNA ISOLATION & cDNA PREPARATION T.ANANTHAKUMAR BMS14312

2. PRINCIPLE The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components.

3. Cont.. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases . RNA remains only in the aqueous phase. After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But the DNA and proteins can recover by sequential separation after the removal of aqueous phase

4. Cont.. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay, and molecular cloning

5. Note.. RNA will be rapidly degraded by RNases , which are EVERYWHERE, especially on your skin. To prevent RNase contamination, you must wear gloves and use only RNase -free tips and microcentrifuge tubes. All solutions have also been specially treated with agents that deactivate RNase , like diethylpyrocarbonate (DEPC) Be especially careful to keep the RNase -free tips covered when not in use

6. MATERIALS REQUIRED REAGENTS Chloroform (without any additives, such as isoamyl alcohol) Isopropyl alcohol 75% Ethanol (in DEPC-treated water) RNase -free water or 0.5% SDS solution

7. PROCEDURE HOMOGENIZATION Growth medium on the cells was discarded and cells were washed with ice cold 1X PBS The monolayer was then covered with 1 ml of TRIzol and the cells were lysed and homogenized by repeated pipetting.

8. PHASE SEPARATION The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes. 0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL LS Reagent was added. The tubes were shaked vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes.

9. Cont.. The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C). The aqueous phase was transferred to other tubes. Following centrifugation, the mixture separates into a lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase.

10. Cont.. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL LS Reagent used for homogenization.

11. RNA PRECIPITATION The RNA was precipitated from the aqueous phase by mixing with 3 microlitre of glycogen and 500 microlitre of isopropyl alcohol . The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C). ( The RNA precipitate forms a gel-like pellet on the side of the tube at bottom).

12. RNA WASH The supernatant was removed. The RNA pellet was washed once with 75% ethanol, adding 900 microlitre of 75% ethanol per 0.75 ml of TRIZOL LS Reagent used for the initial homogenization. The sample were inverted and mixed and centrifuged at 12,000 rpm for 30 minutes at 4 degree.

13. REDISSOLVING RNA RNA was dissolved in RNase -free water (or 0.5% SDS solution) by passing the solution through the pipette tip for a few times, and incubating for 10 minutes at 55 to 60°C.

14. c DNA PREPARATION cDNA is created from a mature mRNA from a eukaryotic cell with the use of an enzyme known as reverse transcriptase. In eukaryotes, a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from tRNA and rRNA and can therefore be used as a primer site for reverse transcription

15. m RNA EXTRACTION Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for purifying RNA such as trizol extraction and column purification . Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind.

16. Cont.. The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT Purification can be performed by binding mRNAs on a solid matrix to which short strings of thymidylate residues are attached ( oligo dT matrix). The mRNAs are removed again by washing in a low salt buffer .

17. c DNA CONSTRUCTION Once mRNA is purified , oligo-dT is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can be extended by reverse transcriptase to create the complementary DNA strand . Now, the mRNA is removed by using an RNAse enzyme leaving a single stranded cDNA ( sscDNA ). This sscDNA is converted into a double stranded DNA with the help of DNA polymerase.

18. Cont … However, for DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed. This is provided by the sscDNA itself by generating a hair pin loop at the 3' end by coiling on it. The polymerase extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action of S1 nuclease.

19. USES OF CDNA LIBRARY cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are used to express eukaryotic genes in prokaryotes. Prokaryotes do not have introns in their DNA and therefore do not possess any enzymes that can cut it out during transcription process. cDNA do not have introns and therefore can be expressed in prokaryotic cells.

20. CONT… Also, it is useful for subsequently isolating the gene that codes for that mRNA. Discovery of novel genes. Clonning of full length cDNA molecules for in vitro study of gene function. Study of the repertoire if mRNAs expressed in different cells or tissues.