TITLE: RNA Extraction for RIN and DV 200 Analysis
AUTHORS: Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark de Caestecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope: Extract RNA for RIN and DV 200 Assessment.</div><div class = "text-block">Expected Outcome:  RIN and DV 200 measurements for tissue quality assessment.</div></div>

[STEPS]
?. [Homogenize Tissue]
Place tissue section in 750 μL of TRIzoILS.
?. [Homogenize Tissue]
Homogenize the sample:Use a homogenizer or  pass sample through a 23G then a 27G needle syringe.
?. [Homogenize Tissue]
Freeze samples on dry ice and re-thaw once.  Mix well by vortexing.
?. [Homogenize Tissue]
Add RNase free water to a final volume of 960 μL.
?. [Homogenize Tissue]
Add  40 μL of glycogen solution (5mg/mL, making a final conc of 200 μg/mL of glycogen) to each tube and mix well.
?. [Collection]
Centrifuge Phase Lock Gel to move gel to the bottom of column (12,000 x g for 5 minutes).Then transfer the TRIzol mixture mixture to the column.
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Incubate for 5 minutes at 20oC (room temperature).
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Add 250 μL of chloroform and shake for 15 seconds.
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Centrifuge at 12,000 x g for 10 minutes at 4oC.
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Move supernatant to a new 1.5 mL tube.
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Add 600  μL isopropanol.
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Vortex and incubate the tube at -20oC for 20 minutes.
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Centrifuge at 20,000 x g for 20 minutes at 4oC.
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Discard supernatant and add 600  μL 80% Ethanol.
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Add 300 μLof RLT plus buffer (from Qiagen RNeasy Plus Micro Kit) containing 2-ME (10 μL of 2-ME for 1 mL of RLT Plus Buffer, 1% 2-ME) and mix.
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Vortex and centrifuge at 20,000 x g for 5 minutes at 4oC
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Discard supernatant and air dry for a few minutes.
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Add 450 μL 100% ethanol (1.5 volumes) and mix well.
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Transfer 750 μLof the sample to RNeasy MinElute spin column and centrifuge for 1 min at 12,000 x g at 24oC.
?. [Collection]
Transfer the collected flow through back through the same column and centrifuge for 1 min at 12,000 x g at 24oC. (This is a double application of sample).  This time discard the flow.
?. [Collection]
Add 500 μL of RPE buffer to the column and centrifuge at 12,000 x g for 1 min at 24oC.
?. [Collection]
Discard flow through and add 500 μL RPE buffer to the column.  Centrifuge at 12,000 x g for 2 min at 24oC.
?. [Collection]
Transfer column to a new collection tube.  Discard the old collection tube with the flow through.
?. [Collection]
Open the lid of the column and centrifuge at 12,000 x g for 5 min at 24oC  to dry up the column membrane completely.
?. [Collection]
Place the column in a new 1.5 mL tube and add 10 uL of RNase free water on the center of the column membrane.  Incubate for 2 min at 20oC.
?. [Collection]
Centrifuge at 12,000 x g for 3 min at 24oC.
?. [Collection]
Add 10 uL of RNase free water again on the center of the column membrane and incubate for 2 min at 20oC.
?. [Collection]
Centrifuge at 12,000 x g for 3 min at 24oC (this is a double elution).
?. [Collection]
Total volume should be ~20 uL.
?. [Collection]
Provide VANTAGE Core with 10 uL of solution for RIN and DV 200 analysis.