Monday, February 11, 2013

Viral RNA extraction: Kit method


RNA extraction by Kit Method:

Here, I am describing the process of viral RNA extraction from QIAamp® Viral RNA extraction kit manufactured by QIAGEN®. There are so many kits available in the market for different purpose. But most of the protocols are somewhat similar in kind. If you know the procedure, you can similarly perform for others too.

Principle
QIAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA for reliable use in amplification technologies. Viral RNA can be purified from plasma (treated with anticoagulants other than heparin), serum, and other cell-free body fluids. Samples may be fresh or frozen, but if frozen, should not be thawed more than once. Repeated freeze–thawing of plasma samples will lead to reduced viral titers and should be avoided for optimal sensitivity. Cryoprecipitates accumulate when samples are subjected to repeated freeze–thawing cycles. This may lead to clogging of the QIAamp membrane when using the vacuum protocol. QIAamp Viral RNA Mini Kits are general purpose kits which can be used for isolation of viral RNA from a wide variety of viruses, but performance cannot be guaranteed for every virus.
Procedure
QIAamp Viral RNA Mini Kits represent a well-established general-purpose technology for viral RNA preparation. The kit combines the selective binding properties of a silicagel-based membrane with the speed of microspin or vacuum technology and is ideally suited for simultaneous processing of multiple samples. QIAamp Viral RNA spin protocols can be fully automated on the QIAcube®. The sample is first lysed under highly denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions are then adjusted to provide optimum binding of the RNA to the QIAamp membrane, and the sample is loaded onto the QIAamp Mini spin column. The RNA binds to the membrane, and contaminants are efficiently washed away in two steps using two different wash buffers. High-quality RNA is eluted in a special RNase-free buffer, ready for direct use or safe storage. The purified RNA is free of protein, nucleases, and other contaminants and inhibitors. The special QIAamp membrane guarantees extremely high recovery of pure, intact RNA in just twenty minutes without the use of phenol/chloroform extraction or alcohol precipitation. All buffers and reagents are guaranteed to be RNase-free.

Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.
  • Ethanol (96–100%)*
  • 1.5 ml microcentrifuge tubes
  • Sterile, RNase-free pipet tips (pipet tips with aerosol barriers for preventing cross contamination are recommended)
  • Microcentrifuge (with rotor for 1.5 ml and 2 ml tubes)


Preparation of reagents:

Addition of carrier RNA to Buffer AVL*
Add 310 μl Buffer AVE to the tube containing 310 μg lyophilized carrier RNA to obtain a solution of 1 μg/μl. Dissolve the carrier RNA thoroughly, divide it into conveniently sized aliquots, and store it at –20°C. Do not freeze–thaw the aliquots of carrier RNA more than 3 times. Check Buffer AVL for precipitate, and if necessary incubate at 80°C until the precipitate is dissolved. Calculate the volume of Buffer AVL–carrier RNA mix needed per batch of samples by selecting the number of samples to be simultaneously processed from Table 1. For larger numbers of samples, volumes can be calculated using the following sample calculation:

n x 0.56 ml = y ml
y ml x 10 μl/ml = z μl

where: n = number of samples to be processed simultaneously
y = calculated volume of Buffer AVL
z = volume of carrier RNA–Buffer AVE to add to Buffer AVL

Gently mix by inverting the tube 10 times. To avoid foaming, do not vortex.

Buffer AW1
Buffer AW1 is supplied as a concentrate. Before using for the first time, add the appropriate amount of ethanol (96–100%) as indicated on the bottle (For 50 tubes, it’s 25 ml , final volume 44 ml). Buffer AW1 is stable for 1 year when stored closed at room temperature, but only until the kit expiration date.

Buffer AW2
Buffer AW2 is supplied as a concentrate. Before using for the first time, add the appropriate amount of ethanol (96–100%) to Buffer AW2 concentrate as indicated on the bottle (For 50 tubes, it’s 30ml and final volume will be 43ml). Buffer AW2 is stable for 1 year when stored closed at room temperature, but only until the kit expiration date.

Handling of QIAamp Mini columns
Owing to the sensitivity of nucleic acid amplification technologies, the following precautions are necessary when handling QIAamp Mini columns to avoid cross contamination between sample preparations:
  1. Carefully apply the sample or solution to the QIAamp Mini column.
  2. Pipet the sample into the QIAamp Mini column without wetting the rim of the column.
  3. Change pipet tips between all liquid transfer steps. The use of aerosol-barrier tips is recommended.
  4. Avoid touching the QIAamp membrane with the pipet tip.
  5. After all pulse-vortexing steps, briefly centrifuge 1.5 ml microcentrifuge tubes to remove drops from the inside of the lid.
  6. Wear gloves throughout the procedure. In case of contact between gloves and sample, change gloves immediately.

Fig. Molecular Diagnostic Lab (MDL), PAHS
Protocol: Purification of Viral RNA (Spin Protocol)
This protocol is for purification of viral RNA from 140 μl plasma, serum, urine, cell culture media, or cell-free body fluids using a microcentrifuge. For automated purification of viral RNA using the QIAamp Viral RNA Mini Kit on the QIAcube, refer to the QIAcube User Manual and the relevant protocol sheet. Larger starting volumes, up to 560 μl (in multiples of 140 μl), can be processed by increasing the initial volumes proportionally and loading the QIAamp Mini column multiple times, as described below in the protocol. Some samples with very low viral titers should be concentrated before the purification procedure.

Important points before starting
All centrifugation steps are carried out at room temperature (15–25°C).

Things to do before starting
  1. Equilibrate samples to room temperature (15–25°C).
  2. Equilibrate Buffer AVE to room temperature for elution in step 11.
  3. Check that Buffer AW1 and Buffer AW2 have been prepared according to the instructions provided.
  4. Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions provided.

Procedure

1. Pipet 560 μl of prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge tube.
If the sample volume is larger than 140 μl, increase the amount of Buffer AVL–carrier RNA proportionally (e.g., a 280 μl sample will require 1120 μl Buffer AVL–carrier RNA) and use a larger tube.

2. Add 140 μl plasma, serum, urine, cell-culture supernatant, or cell-free body fluid to the Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s.
To ensure efficient lysis, it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution. Frozen samples that have only been thawed once can also be used.

3. Incubate at room temperature (15–25°C) for 10 min.
Viral particle lysis is complete after lysis for 10 min at room temperature. Longer incubation times have no effect on the yield or quality of the purified RNA. Potentially infectious agents and RNases are inactivated in Buffer AVL.

4. Briefly centrifuge the tube to remove drops from the inside of the lid.

5. Add 560 μl of ethanol (96–100%) to the sample, and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.

Only ethanol should be used since other alcohols may result in reduced RNA yield and purity. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. If the sample volume is greater than 140 μl, increase the amount of ethanol proportionally (e.g., a 280 μl sample will require 1120 μl of ethanol). In order to ensure efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution.

6. Carefully apply 630 μl of the solution from step 5 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate.
Close each spin column in order to avoid cross-contamination during centrifugation. Centrifugation is performed at 6000 x g (8000 rpm) in order to limit microcentrifuge noise. Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the solution has not completely passed through the membrane, centrifuge again at a higher speed until all of the solution has passed through.

7. Carefully open the QIAamp Mini column, and repeat step 6.
If the sample volume was greater than 140 μl, repeat this step until all of the lysate has been loaded onto the spin column.

8. Carefully open the QIAamp Mini column, and add 500 μl of Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.
It is not necessary to increase the volume of Buffer AW1 even if the original sample volume was larger than 140 μl.

9. Carefully open the QIAamp Mini column, and add 500 μl of Buffer AW2. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with step 11, or to eliminate any chance of possible Buffer AW2 carryover, perform step 10, and then continue with step 11.

Note: Residual Buffer AW2 in the eluate may cause problems in downstream applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow-through, containing Buffer AW2, contacting the QIAamp Mini column. Removing the QIAamp Mini column and collection tube from the rotor may also cause flow-through to come into contact with the QIAamp Mini column. In these cases, the optional step 10 should be performed.

10. Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not provided), and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.

11. Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not provided). Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 60 μl of Buffer AVE equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min. Centrifuge at 6000 x g (8000 rpm) for 1 min.
A single elution with 60 μl of Buffer AVE is sufficient to elute at least 90% of the viral RNA from the QIAamp Mini column. Performing a double elution using 2 x 40 μl of Buffer AVE will increase yield by up to 10%. Elution with volumes of  less than 30 μl will lead to reduced yields and will not increase the final concentration of RNA in the eluate. Viral RNA is stable for up to one year when stored at –20°C or –70°C.

(Source: QIAamp® Viral RNA Mini Handbook)
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