This document discusses methods for extracting and enriching mRNA from biological samples. It describes acid guanidinium thiocyanate-phenol-chlorform extraction and CsCl gradient purification for total RNA extraction. For eukaryotes, mRNA is enriched using oligo-dT hybridization to the poly-A tail. For prokaryotes which lack a poly-A tail, methods include rRNA capture using probes, degradation of already processed RNAs leaving mRNA, selective polyadenylation of mRNA, and antibody capture of specific mRNAs associated with proteins. Purified mRNA can be used for applications like transcriptome sequencing, gene expression profiling, and techniques like RT-PCR, microarrays, and northern blotting.

1. mRNA Enrichment
Basic Molecular Biology
BIO-NIIST-2-4104
Jasmin G. Russel
10BB15A39017

2. RNA Extraction
• Purification of RNA from biological samples
• Acid Guanidinium thiocyanate-Phenol-
Chloroform extraction (AGPC)
• CsCl gradient purification
Total RNA
80-85% rRNA
15-20% tRNA
1-5% mRNA
– Variable size
– 3’ polyadenylation

3. Organic RNA Extraction
1. Lyse/homogenize cells (4M
Guanidinium thiocyanate, pH 4)
2. Add phenol:chloroform:isoamyl
alcohol to lysed sample, and
centrifuge
3. Organic phase separates from
aqueous phase
– Organic solvents on bottom
– Aqueous phase on top
(contains total RNA)
– Cellular debris and genomic
DNA appears as a “film” of
debris at the interface of the
two solutions
4. Remove RNA solution to a clean
tube; precipitate RNA and wash
with ethanol, then resuspend
RNA in water

4. Affinity Purification of RNA
1. Lyse cells, and spin to remove large particulates/cell
debris
2. Apply lysate (containing nucleic acids and cellular
contaminants) to column with glass membrane
3. Wash with alcohol to remove contaminants; nucleic
acids stick to glass membrane while contaminants
wash through. Treat with DNase enzyme to remove
contaminating DNA.
4. Apply water to the column; purified RNA washes off the
glass and is collected

5. Determining Purity
• Ratio of the readings : O.D.260/O.D.280 is a
measure of purity.
• If the 260nm/280nm ratio is less than 2.0-
2.3 for RNA this indicates contamination,
usually with protein.
• Pure preparations of DNA and RNA have O.D
260/280 of 1.8 and 2.0 respectively.

6. mRNA Enrichment - Eukaryotes
• Poly A tail
• 30-200 nt long
• Hybridisation of Poly A tail containing RNAs
to oligo dT molecules connected to a carrier
– Cellulose matrix
– Biotinylated oligo dT
• Washing of nucleic acids which do not bind
to oligo dT
• Elution of Poly A RNA from carrier

8. mRNA Enrichment Prokaryotes
• Lack long 3’poly A tail
Methods
• rRNA Capture
• Degradation of already processed RNA
• Polyadenylation of mRNA
• Antibody capture of specific RNAs

9. Ribosomal RNA Capture
• Probes correspond to the conserved regions
of 16S and 23S regions
• Probes attached to metallic beads are
removed with rRNA
• Specific for an organism
• Probes differ from organism to organism
• MICROBExpress Kit by Ambion

11. Degradation of processed RNA
• mRNA have 5’triphosphate (5’PPP)
• rRNA and tRNA have 5’monophosphate (5’P)
• Exonuclease – breaks down 5’P RNA
molecules only
• mRNA only prokaryotic mRNA isolation kit
by Epicentre

13. Selective polyadenylation of mRNA
• E. coli poly (A) polymerase to poly adenylate
the mRNA
• After polyadenylation oligo dT probes are
used for capturing
• Message AMP II bacteria kit by Ambion

15. Antibody Capture
• Immunoprecipitation of mRNAs associted
with certain protein
– Hfq, associated with rRNA and tRNA
– Hfq Co-IP

16. Applications
• Transcriptome sequencing
• Gene expression profiling.
• RNA profiling techniques such as
• Real Time RT-PCR
• Microarray analysis
• Northern blotting

17. Thank you