Dec 12, 2016
Total RNA Purification from Plasma or Serum (ISOLATE II Biofluids RNA Kit)
- Bioline
- Bioline
External link: http://www.bioline.com/us/downloads/dl/file/id/3789/isolate_ii_biofluids_rna_kit_product_manual.pdf
Protocol Citation: Bioline 2016. Total RNA Purification from Plasma or Serum (ISOLATE II Biofluids RNA Kit). protocols.io https://dx.doi.org/10.17504/protocols.io.f5hbq36
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 18, 2016
Last Modified: March 08, 2018
Protocol Integer ID: 3977
Abstract
Protocol for RNA Purification form Plasma or Serum, using the ISOLATE II Biofluids RNA Kit. This protocol includes the lysate preparation procedure.
Guidelines
Before you start:
Plasma or serum of all human and animal subjects is considered potentially infectious. All
necessary precautions recommended by the appropriate authorities in the country of use
should be taken when working with plasma or serum.
We recommend the use of this kit to isolate RNA from plasma or serum prepared by a
standard protocol from non-coagulated, fresh whole blood using EDTA or sodium citrate as
the anti-coagulant.
Plasma prepared from fresh blood using heparin as an anti-coagulant is not suitable for use
with this protocol.
Due to the relatively low DNA content in plasma, the Genomic DNA Removal Column is not
necessary for this protocol.
It is recommended that no more than 200 μL of plasma or serum is used in order to prevent
clogging of the column.
Yields of RNA from plasma and serum is highly variable. In general, the expected yield
could vary from 1 to 100 ng per 100 μL plasma or serum used. In addition, the expected A260/A280 ratio as well as the A260/A230 ratio will be lower (<1.8) than the normal acceptable range from other cells or tissues. Nonetheless, these isolated RNA can be effectively used in different downstream applications such as real-time PCR or microarrays.
Avoid multiple freeze-thaw cycles of the plasma or serum sample. Aliquot out the appropriate volume for usage prior to freezing.
It is important to work quickly during this procedure.
Please review the Guidelines under Genomic DNA removal and total RNA purification from all types of lysate for other important details.
Materials
MATERIALS
ISOLATE II Biofluids RNA KitBiolineCatalog #BIO-52086
Safety warnings
Plasma or serum of all human and animal subjects is considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with plasma or serum.
When working with chemicals, always wear a suitable lab coat, gloves and safety glasses.
Lysis Buffer RX contains guanidinium thiocyanate. This chemical is harmful in liquid form when in contact with skin or ingested. If the solution is allowed to dry, the powder is harmful if inhaled.
CAUTION: Do not add bleach directly to solutions or sample preparation waste containing guanidinium salts. Reactive compounds and toxic gases can form. In the case of spillage, clean the affected area with a suitable laboratory detergent and water.
For detailed information, please consult the material data safety sheet (MSDS) available on
our website at www.bioline.com.
Biofluids derived from all human and animal sources are considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with biofluids.
Lysate Preparation from Plasma/Serum
Lysate Preparation from Plasma/Serum
Transfer up to 200 μL of plasma or serum to a 1.5mL RNase-free microcentrifuge tube (not supplied).
Add 300 μL of Lysis Buffer RX to every 100 μL of plasma or serum.
Mix by vortexing for 10s.
00:00:10
Optional: Add 0.7μL of 0.8μg/μL MS2 RNA per sample.
Note
Note: The use of MS2 RNA can increase the consistency of downstream applications such as real-time- PCR and RT-PCR. However, the use of MS2 RNA is not recommended for applications involving global gene expression analysis such as microarrays or sequencing.
Add 400 μL of 96-100% ethanol to every 400 μL of lysate (equivalent to every 100μL plasma or serum used).
Mix by vortexing for 10s.
00:00:10
Binding RNA to Column
Binding RNA to Column
Assemble an ISOLATE II RNA Column (black ring) with a provided Collection Tube.
Apply up to 600μL of the ethanolic lysate onto the column and centrifuge for 1 min at ≥ 3,500 x g.
00:01:00
Note
Note: Ensure the entire lysate volume has passed through into the Collection Tube by inspecting the column. If the entire lysate volume has not passed through, spin for an additional minute at 14,000 x g.
Discard the flow-through. Reassemble the spin column with its Collection Tube.
Depending on the lysate volume,repeat steps 8 and 9 as required.
RNA Column Wash
RNA Column Wash
Apply 400 μL of 96-100% ethanol to the column and centrifuge for 1 min at 14,000 x g. (wash 1/3)
00:01:00
Note
Note: Ensure the entire wash buffer volume has passed through into the Collection Tube by inspecting the column. If the entire wash buffer volume has not passed through, spin for an additional minute at 14,000 x g.
Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 1/3)
Apply 400 μL of 96-100% ethanol to the column and centrifuge for 1 min at 14,000 x g. (wash 2/3)
00:01:00
Note
Note: Ensure the entire wash buffer volume has passed through into the Collection Tube by inspecting the column. If the entire wash buffer volume has not passed through, spin for an additional minute at 14,000 x g.
Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 2/3)
Wash column a third time by adding 400μL of 96-100% ethanol and centrifuge for 1 min
at 14,000 x g. (wash 3/3)
00:01:00
Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 3/3)
Spin the column for 2 min at 14,000 x g in order to dry the column thoroughly. Discard the
Collection Tube.
00:02:00
RNA Elution
RNA Elution
Place the column into a fresh 1.7mL Elution Tube.
Add 50μL of RNA Elution Buffer to the column.
Centrifuge for 2 min at 200 x g.
00:02:00
Centrifuge for 1 min at 14,000 x g. Note the volume eluted from the column. If the entire 50 μL has not been eluted, spin the column at 14,000 x g for an additional minute to elute the RNA.
00:01:00
Note
Note: For maximum RNA recovery, it is recommended to apply a second volume of RNA Elution Buffer and elute into the same microcentrifuge tube (repeat steps 19-21). Alternatively, re-apply the first eluate onto the column and re-elute into the same microcentrifuge tube (for high concentration).
Storage of RNA
Storage of RNA
The isolated RNA can be stored at -20°C for a few days or at -80°C (recommended) for long-term storage.