InCellGene^TM Blood Total RNA Miniprep Kit

Cat.No.:IC-5300

Introduction

The InCellGene^TM Blood Total RNA Miniprep Kit provides an easy and fast method for isolating total RNA from white blood cells within 30 min. The kit combines the reversible binding properties of ezBind RNA technology with a specially designed buffer system which can effectively remove DNA before RNA isolation.The lysate is passed through an InCellGene™ DNA Clearance Column which will trap the genomic DNA. And trace genomic DNA can be eliminated by DNase I treatment (See detail in the protocol) when it is necessary.

Storage and Stability

All components can be stored at room temperature (15-25¡æ). All kit components are guaranteed for 12 months from the date of purchasing.

Kit Contents

Before Starting

Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step.

Important

1.Add 1% volume of ¦Â-mercaptoethanol to Buffer LY before use and store at 4¡æ.

2.Add 8 mL (R6411-00) or 96 mL (R6411-01) or 96 mL (R6411-02) 100%ethanol to each RNA Wash Buffer before use.

3.Red Blood Cell Lysis Solution is supplied as 10 x concentrate. Calculate the amount of buffer to be used and mix one part of 10 x Red Blood Cell Lysis Solution with 9 part of ddH2O before use.

Materials supplied by users

1.Tabletop microcentrifuge .

2.Vacuum manifold if use vacuum protocol.

3.100% ethanol

Note: Perform all steps including centrifugation at room temperature.

Protocol for Total RNA Extraction From White Blood Cells (Leukocytes)

Calculate the amount of 10 x Red Blood Cell Lysis Solution to be used. Mix one part of 10 x Red Blood Cell Lysis Solution with 9 part of ddH₂O before use.

1.Transfer 1 mL of whole blood (collected in heparinized or EDTA-treated tubes) into a 15 mL conical tube. Add 3 volumes of Red Blood Cell Lysis Solution and mix the solution by inversing the tube 5 times. Incubate on ice for 10 min.

2.Centrifuge the blood sample at 3,000 rpm (600 x g) for 5 min at 4 ¡æ. Remove the supernatant by carefully pipetting from the top of the sample. Do not disturb the precipitated leukocyte pellets. Carefully wash the pellets with 2 mL of Red Blood Cell Lysis Solution and centrifuge the sample at 3,000 rpm (600 xg) for 5 min at 4^oC.

3.Remove the supernatant without disturbing the leukocyte pellets.

4.Add 500 ¦ÌL Buffer LY to the leukocytes pellets and vortex the solution for 1 min.

5.Transfer the cleared lysate to a DNA Clearance Column pre-inserted in a 2 mL Collection Tube. Centrifuge at 13,000 rpm for 2 min. Discard the DNA Clearance Column and save the flow-through.

Note£ºThis step is for genomic DNA removal.

6.Add 0.5 volume of 100% ethanol to the lysate (for example: 250 ¦ÌL 100% ethanol for 500 ¦ÌL lysate).

7.Transfer the solution into a RNA Column and centrifuge at 13,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.

8.Add 500 ¦ÌL Buffer RB to the column and centrifuge at 13,000 rpm for 30s.

Discard the flow-through.

9.Add 500 ¦ÌL RNA Wash Buffer to the column and centrifuge at 13,000 rpm for 1 min. Discard the flow-through.

Ensure that ethanol has been added to RNA Wash Buffer before use.

10.Add another 500 ¦ÌL RNA Wash Buffer to the column and centrifuge at  14,000 rpm for 30 s. Discard the flow-through.

11.Centrifuge the column at 13,000 rpm for 1 min. Discard the flow-through and collection tube.

12.Put the column into a new collection tube. Centrifuge the column, with the lid open, at 13,000 rpm for 2 min.

It is critical to remove residual ethanol for optimal elution.

13.Place the column to a 1.5 mL RNase-free Microfuge Tube, add 50-100 ¦ÌL DEPC-treated ddH2O to the column and centrifuge at 13,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution at -20¡æ.

Note: It is highly recommended that RNA quality be determined before downstream applications. The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining. Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations  of mRNA and bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling or storage, RNA molecule less than 200 bases in length do not efficiently bind to the RNA Column. An A260/A280 ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.