Blood Total RNA Kit

Product Composition

Product Storage and Expiry Date

1.       Buffer L9 can be transported at room temperature, please store at 2~8 ℃ after receiving the product.

2.       Other reagents and components are stored at room temperature (0~30 ℃), the performance of the product can be maintained without significant change within two years; if the product is stored at 2~8 ℃, the validity period of the product can be extended to more than two years (the product stored at 2~8℃ should be restored to room temperature before use).

Product Description

This product is suitable for the isolation of total RNA (including viral RNA from whole blood) from 500 µl fresh or frozen whole blood or bone marrow (frozen samples must have been frozen at -70 ℃ for less than one year and have not been thawed during that time). Samples are lysed in strong lysis buffer, hemoglobin and genomic DNA are removed in the form of precipitate. RNA can be bound to spin column after adding ethanol to the supernatant and the dissolved protein and PCR inhibitor are filtered out. Add Buffer WA and Buffer WBR to wash the spin column, and purified RNA is eluted with RNase-free Water. The purified RNA is ready for use in RT-PCR, Northern blot, Dot blot and other molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1． Absolute ethanol

2． 1.5 ml microcentrifuge tubes (RNase-free), 2 ml microcentrifuge tubes

3． pipettes and tips (RNase-free pipette tips with filter cartridges are recommended to avoid RNase contamination)

4． Protective equipment such as disposable latex gloves and paper towels

5．Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)

6． Vortexer

7．Laboratories that do not use RNase

Preparation Before Use

1.       If the centrifuge has refrigeration function, please set the temperature to 25°C.

2.       Add absolute ethanol to Buffer WA and Buffer WBR according to the instructions on the label of the reagent bottle, and tick the box on the label to mark "Ethanol added".

3.       Since saliva and skin contain RNase, disposable gloves and masks are required during the whole process of RNA extraction.

4.       It is best to use fresh whole blood or bone marrow within 3 hours in vitro for RNA extraction, otherwise the final recovery efficiency of RNA will be affected due to the degradation of RNA. If the fresh sample cannot be extracted in time for RNA, the sample can be frozen at -70℃ or frozen at -20℃ after mix with Buffer L9. (See Step 1 for details)

Protocol:

1.      Add 1 ml Buffer L9 to a 2 ml microcentrifuge tube, add 500 µl whole blood or bone marrow and mix well by vortexing for 30 seconds.

* If fresh whole blood or bone marrow cannot be extracted for RNA in time, the sample can be frozen at -70℃. Samples frozen for less than one year will not affect the extraction of RNA. It should be noted that the samples should not be frozen and thawed repeatedly before RNA extraction.

* If there is no -70 ℃ refrigerator, the lysed sample can be frozen at -20 ℃ after this step. Freezing for two weeks does not affect the efficiency of RNA extraction.

* Buffer L9 is corrosive, please wear protective equipment for operation.

2.      Centrifuge at 13000 rpm for 10 minutes. Add 500 μl absolute ethanol to a clean 1.5 ml microcentrifuge tube.

3.      Pipette 700 µl supernatant into the 1.5 ml microcentrifuge tube with absolute ethanol and mix by pipetting twice directly.

* The hemoglobin contained in the supernatant can be removed during the washing step without affecting the final RNA purification effect.

4.      Pipette 600 µl mixture from step 3 to a spin column (the column is placed in a 2 ml collection tube), close the lid and centrifuge at 12000 rpm for 30 seconds.

5.      Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, pour all the remaining liquid in the 1.5 ml microcentrifuge tube into the spin column, close the lid and centrifuge at 12000 rpm for 30 seconds.

* The filtrate does not need to be completely discarded, if you want to avoid the contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can slap the 2 ml collection tube upside down on a paper towel once.

6.      Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 500 µl Buffer WA to the spin column, close the lid and centrifuge at 12000 rpm for 30 seconds.

* Make sure absolute ethanol has been added into Buffer WA.

7.      Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 700 µl Buffer WBR to the spin column, close the lid and centrifuge at 12000 rpm for 30 seconds.

* Make sure absolute ethanol has been added into Buffer WBR.

8.      Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube and centrifuge at 14000 rpm for 1 minute.

* If the top speed could not reach 14000 rpm, centrifuge at full speed for 2 minutes.

* Do not omit this step, otherwise, it may cause problems in downstream applications due to the residual ethanol in the eluate.

9.      Discard the 2 ml collection tube and place the spin column in an RNase-free 1.5 ml microcentrifuge tube, add 50 µl RNase-free Water to the spin column, close the lid and incubate for 1 minute at room temperature, then centrifuge at 12000 rpm for 30 seconds.

* If the centrifuge does not have a leak-proof cap, please change the centrifugation condition to 8000 rpm for 1 minute to prevent the 1.5 ml microcentrifuge tube cover from falling off and damaging the centrifuge.

10.   Discard the spin column and the eluted RNA can be used immediately for various molecular biology experiments; store the RNA below -70℃ for later use.

* Even if no DNA band is detected by gel electrophoresis, it does not mean that there is no DNA in the eluted RNA. If you want to remove DNA completely, digest the residual DNA with DNase I.

* If it is used for viral RNA detection, the sensitivity of detection can be improved by increasing the amount of template (25 µl eluted RNA was added to the one-step RT-qPCR reaction system with a final volume of 50 µl as the template, and no significant inhibitory effect was observed).