TITLE: RNA Extraction
AUTHORS: Alicia Rich

[STEPS]
?. [Lyse and Homogenize]
Do not allow samples to thaw before adding the . If a sample needs to be thawed for measuring, do so on ice.
?. [Lyse and Homogenize]
Use sterilized forceps to transfer swab or ~90 mg of feces into a 2 ml microcentrifuge tube held on ice or in a cooling block.
When processing samples containing average or low amounts of RNA, the maximum amount of starting material shown in Table 1 (kit manual) can be used. However, even though the RNA binding capacity of the RNeasy spin column is not reached, the maximum amount of starting material must not be exceeded. Otherwise, lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane, resulting in lower RNA yield and purity.
?. [Lyse and Homogenize]
For feces: add  (or as much as you can filling the tube).For swab: add .
1.8 mL
600 µl
?. [Lyse and Homogenize]
Place the tubes in the shaking block at maximum speed and minimum temperature for 20 minutes.
4 33
?. [Lyse and Homogenize]
For feces: transfer   of the lysate into a  tube. Keep the rest of the lysate in the original tube on ice.For swab: transfer the entire lysate volume and swab tip into a  tube.
700 µl
?. [Lyse and Homogenize]
Spin the column at the maximum speed for 2 minutes in the centrifuge at the extraction station.
Centrifuge: 10000 33, 2 min
?. [Lyse and Homogenize]
For feces only:
[Repeat until you have used all of the lysate or filled the 2 ml tube.]
?. [Lyse and Homogenize]
For feces only: Transfer the lysate into another 2 ml microcentrifuge tube, and replace the same one with the same spin column.
?. [Lyse and Homogenize]
For all samples: Centrifuge the total lysate volume for 3 min at 10,000 rpm.
Centrifuge: 10000 33, 3 min
?. [Remove genomic DNA]
Carefully remove the supernatant by pipetting, and transfer it to a  gDNA Eliminator spin column placed in a 2 ml collection tube (included in the kit).
?. [Remove genomic DNA]
Centrifuge for 30 s at 10,000 rpm. Discard the column and save the flow-through.
Centrifuge: 10000 33, 30 s
?. [Remove genomic DNA]
Make sure that no liquid remains on the column membrane after centrifugation. If necessary, repeat this step until all liquid has passed through.
?. [Remove genomic DNA]
Add   50% ethanol to the flow-through, and mix well by pipetting. DO NOT CENTRIFUGE. Work quickly and proceed immediately to next step.
[(or equal volume)]
If some lysate was lost during the homogenization and DNA removal, adjust the volume of ethanol accordingly.Precipitates may be visible after the addition of ethanol, but this will not affect the procedure.
?. [Bind the RNA]
Transfer up to  of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (included in the kit). Keep any remaining sample in the same tube on ice.
700 µl
?. [Bind the RNA]
Centrifuge for 15 s  at 10,000 rpm or faster.  Discard the flow-through, but save the tube for the next step.
Centrifuge: 10000 33, 15 s
?. [Bind the RNA]
If any sample remained in the tube, repeat until all liquid has passed through the column.
?. [Wash]
Add  to the RNeasy spin column with the same empty collection tube from the previous step. Close the lid gently.
700 µl
?. [Wash]
Centrifuge for 15 s at 10,000 rpm or faster to wash the column membrane. Discard the flow-through.
Centrifuge: 10000 33, 15 s
After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.
?. [Wash]
Add  to the RNeasy spin column with a fresh collection tube. Close the lid gently.
500 µl
Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
?. [Wash]
Centrifuge for 15 s at 10,000 rpm for faster. Discard the flow-through, but save the collection tube again for the next step.
Centrifuge: 10000 33, 15 s
?. [Wash]
Add  to the RNeasy spin column with the same emptied collection tube from the previous step. Close the lid gently.
500 µl
?. [Wash]
Centrifuge for 2 min at 10,000 rpm or faster.
Centrifuge: 10000 33, 2 min
Carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, a carryover of ethanol will occur.
?. [Wash]
Place the RNeasy spin column in a new 2 ml collection tube, and discard the old collection tube with the flow-through. Centrifuge at full speed for 1 min.
Centrifuge: 10000 33, 1 min
?. [Elute]
Place the RNeasy spin column in a new 1.5 ml collection tube, clearly labeled with the sample ID, date, and your initials. Add directly to the spin column membrane. Close the lid gently, and allow the tube to sit on ice for 10 minutes.
70 µl
?. [Elute]
Centrifuge for 1 min at 10,000 rpm to elute the RNA. DO NOT DISCARD THE FLOW-THROUGH OR COLUMN.
Centrifuge: 10000 33, 1 min
?. [Elute]
[with a second labeled collection tube and the same column.]
?. [Elute]
Immediately transfer both tubes to the freezer.   ( if you plan to carry out PCR immediately)
-80 °C
-20 °C