RNA ISOLATION

Madhumitha, Project Assistant, MDRF

RNA- An Introduction

RNA- build into chain with the nucleotides

Adenine Guanine Cytosine Uracil

Differences b/w RNA & DNA Single stranded Thymine is replaced by Uracil Ribose sugar

Types- mRNA, rRNA, tRNA

RNA ISOLATION- MATERIALS REQUIRED

Sample (whole blood, tissue, cell lines) Ficoll Histopaque Trizol Chloroform Iso-Propyl alcohol RNAse free std lab wares

Trizol- Guanidium Isothiocynate Inactivation of RNAse Maintains RNA stablitiy Break down cells and release the cell

components Chloroform

Partitioning of RNA in aqueous phase Precipitation of proteins

Isopropyl alcohol Facilitates RNA precipitation

RNA ISOLATION- STEPS INVOLVED Ficoll Histopaque separation of

Lymphocytes 2ml ficoll + layer 2ml of whole blood Centrifuge at 1000 rpm for 30 mins

Ficoll

Transfer the buffy coat to microfuge tube and centrifuge again at 10,000 rpm for 10 mins

Pellet+ 1ml of Trizol Incubate at RT for 5 mins 200 ul of chloroform (1/5 vol of trizol) Incubate at RT for 10 mins Cetrifuge- 10,000 rpm for 10 mins

Tranfer the supernatant to a fresh tube and add equal volume of Iso-propyl alcohol

RNA is precipitated Air dry and dissolve 50-100 ul of RNAse

free water

Applications

cDNA Synthesis Real Time PCR Gene expression studies

mRNA profiling Micro RNA profiling

THANK YOU