RNA Extraction-Dr upasana ray class .pdf

RNA Extraction

1. RNA Extraction and RNA Handling
Dr. UPASANA RAY
SENIOR SCIENTIST
DIVISION: INFECTIOUS DISEASES AND IMMUNOLOGY
CSIR-INDIAN INSTITUTE OF CHEMICAL BIOLOGY
Kolkata, West Bengal

3. Reverse transcription

6. Virus Life Cycle

7. Diagnostic Methods for Viral Infections

9. Testing approaches
Nucleic-acid tests directly probe
for the RNA of viruses swabbed
from a patient’s throat or nasal
passage
Serological tests detect
antibodies present in
the patient’s serum
Antigen tests: Detect
viral proteins present in
the patient’s body fluid
IgG and IgM antibody production, termed seroconversion, typically occurs 5-10 days after the onset of
initial symptoms (Wölfelet al. 2020).
Hence, nucleic acid tests offer the earliest and most sensitive detection for the presence of SARS CoV-2

10. How is viral nucleic acid detected?
Quantitative real time reverse transcriptase-polymerase chain reaction
i.e. RT-PCR
The Gold Standard for clinical diagnosis and confirmation of SARS-CoV-2
infection (and also applicable for other viral infections)

11. Sample
RNA extraction
RT-PCR

12. A clinician collects a nasopharyngeal swab and transfers it to a
vial containing viral transport medium (VTM), which is then
transported to laboratory for testing.

13. Handling RNA
KEEP WORK SPACE AND REAGENTS CLEAN

14. RNase is a nuclease, an enzyme that breaks down RNA into smaller units
RNases can be of two types: endoribonucleasesandexoribonucleases
Ribonucleases/RNases

15. Handling RNA
•Ribonucleases (RNases) are very stable and active enzymes that generally do not require
cofactors to function.
•Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA

16. Always use disposable gloves, change frequently and keep
laboratory dust free
Always use RNase free tubes and tips. Never keep tubes and containers open
Handling RNA

17. Handling RNA
•Do not use any plasticware or glassware without first eliminating possible RNase
contamination.
•Great care should be taken to avoid inadvertently introducing RNases into the
RNA sample during or after the purification procedure.

18. DEPC treatment of solutions (other than kit
contents)and Glassware
Solutions (water and other solutions) should be treated with 0.1% DEPC. {Nuclease free
water may also be used}
DEPC is a strong inhibitor of RNases.
DEPC inactivates RNases by covalent modification.
Treatment of water by DEPC
•Add 0.1 ml DEPC to 100 ml of the solution to be treated
•Shake vigorously to bring the DEPC into solution
•Let the solution incubate for 12 h at 37°C
•Autoclave for 15 min to remove any trace of DEPC
•When preparing buffers, treat water with DEPC first, and then make the appropriate
buffer.
Diethyl pyrocarbonate (DEPC)

19. RNA isolation

20. All you need….
The kit/reagents
96-100% Ethanol
Set of pipette
Vortex mixer
Centrifuge
Biosafety cabinet/ facility and PPE

21. •The kit combines the selective binding properties of a silica-based membrane with the speed of microspinor
vacuum technology
•It is highly suited for simultaneous processing of multiple samples.
The Kit
Spin columns and
collection tubes
Buffer AVL
Buffers AW1 and AW2
Carrier RNA
Buffer AVE

22. Time taken for RNA purification: 20-30 minutes as per manufacturer’s protocol
(Traditional phenol-Chloroform and alcohol based methods take much longer time)

23. The four basic steps of nucleic acid
extraction using columns

24. RNA Extraction columns available with the kit

25. Composition of sample
Nucleic acids
Protein
Nucleases Other contaminants

26. What do we need?
Break the virus
particle
Isolate the RNA

27. The sample is first lysedunder
highly denaturing conditions to
inactivate RNases and to ensure
isolation of intact viral RNA.
The sample is loadedonto
the QIAampMini spin column
The RNA binds to the membrane,
and contaminants are efficiently
washedaway in 2 steps using 2
different wash buffers
RNA is elutedin a special RNase-
free buffer, ready for direct use or
safe storage
The major steps
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28. Lysis and Membrane binding
The sample is first lysed under the highly denaturing conditions toinactivate RNasesand to
ensure isolation of intact viral RNA.
Carrier RNA, added to lysis buffer, improves the binding of viral RNA to the membrane of the
extraction column especially in the case of low-titer samples, and limits possible degradation
of the viral RNA due to any residual RNase activity.
Nucleic acidsbinds to the
silica membrane in the
presence of buffer of high
ionic strength

29. Carrier RNA
Carrier RNA plays two important roles:
1. It enhances binding of viral nucleic acids to the column membrane, especially if there are
very few target molecules in the sample.
2. The addition of large amounts of carrier RNA reduces the chance of viral RNA degradation
in the rare event that RNase molecules escape denaturation by the chaotropic salts and
detergent in Buffer AVL.
If carrier RNA is not added to Buffer this may lead to reduced viral RNA recovery.

30. Two washes have been recommended to help the bound RNA be free of contaminants.
The use of 2 different wash buffers, AW1 and AW2, significantly improves the purity of the
eluted RNA.
Wash
Flow through is
discarded after each
wash
RNA gets bound here on the
membrane

31. Elution with buffer AVE
•AVE is RNase-free water containing 0.04% sodium azide(to prevent microbial growth
and subsequent contamination with Rnases)
•Sodium azideaffects spectrophotometric absorbance readings between 220 and
280 nm but has no effect on downstream applications, such as RT-PCR.
•Elution of RNA can also be done with RNase-free water
Prepared RNA is good to go for qRT-PCR as mentioned in the ‘ICMR-NIV: First line screening
protocol for E gene’

32. Summary of RNA extraction steps

33. Thank you