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RNA miniprep protocol - Tissue/cell

1.       For cells:

a.      For total RNA extraction from suspension cultured cells, collect cells by centrifuging at 300 x g for 5 min. Wash the cell pellet by 4oC 1xPBS and centrifuge at 300 x g for 5 min. Discard the supernatant.
b.      For total RNA extraction from adherent cultured cells, remove the culture medium. Wash the cell by 4oC 1xPBS and remove the PBS. Or following the regular protocol to collect the cells. 
c.       Add 500 µl Buffer LY (add b-mercaptoethanol before use) to the cell pellet or directly into the well (for adherent cells) according to the Table 1.
For tissues:
Quickly weigh an appropriate tissue mass according to Table 1 and transfer the tissue into a 1.5 ml tube. Add 500 µl Buffer LY (add b-mercaptoethanol before use) and homogenize the tissue by a rotor starter or an ultrasonic homogenizer on ice.
 
2.       Homogenize the lysate by vortexing vigorously or repeated pipetting until the sample is uniformly homogenized. If the solution is clear, go to step 5, otherwise go to step 4.
 
3.       Centrifuge the solution at 13,000 rpm for 2 min and transfer the clear lysate to a clean 1.5 mL tube.
 
4.       Add 1/2 volume 96-100% ethanolinto the lysate (For example: 250 µL 96-100% ethanol for 500 µL lysate)and pipet 5 times to mix the solution. Vortex briefly if any precipitations.
 
5.       Transfer the solution to a RNA column and centrifuge at 13,000 rpm for 1 min. Discard the flow-through and put the column back to the collection tube.
 
Without DNase I treatment:
6.       Add 400 µL Buffer RBto the column and centrifuge at 13,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.
 
7.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Repeat once. Ensure that ethanol has been added to RNA wash buffer before use.
 
8.       Centrifuge the column with the lid open at 13,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.
9.       Transfer the column to an RNase-free 1.5 mL tube. Add 50 µL DEPC-treated ddH2O to the center of the column. Centrifuge at 13,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.
 
With DNase I treatment:
6.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use.
 
7.       Add 75 µL DNase I (5URNase-free) with Digestion Buffer onto the middle of the column and incubate at room temperature for 15 min. Add 400 µL Buffer RB to the column, incubate for 1-2 min, and centrifuge at 13,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.
 
8.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Repeat once. Ensure that ethanol has been added to RNA wash buffer before use.
 
9.       Centrifuge the column with the lid open at 13,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.
 
10.   Transfer the column to an RNase-free 1.5 mL tube. Add 50 µL DEPC-treated ddH2O to the center of the column. Centrifuge at 13,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.
 
 Table 1. Recommended Amounts of Tissue and Cells for Preparation

Sample
500 μL Buffer LY
Total RNA Yield (μg)
Liver 
10 mg
50 (10 mg tissue)
Kidney
10 mg
20-30 (10 mg tissue)
Muscle*
10 mg
20 (10 mg tissue)
Spleen
10 mg
30-40 (10 mg tissue)
Heart*
10 mg
50 (10 mg tissue)
Brain**
10 mg
80 (10 mg tissue)
Lung
10 mg
10-20 (10 mg tissue)
Pancreas
10 mg
20 (10 mg tissue)
HeLa Cells
1x106
15 (1x106 cells)
293HEK
1x106
12 (1x106 cells)
COS-7
1 x106
30 (1x106 cells)
NIH/3T3
1x106
10 (1x106 cells)

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/A280ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.
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