C-RNA Sample Preparation Protocol

I. List of Reagents

II. Protocol

Extract Circulating Nucleic Acid from plasma using QIAamp Circulating Nucleic Acid kit

Extract circulating nuclear acid from 2-5 mLs of plasma using the instructions provided in the QIAamp circulating nucleic acid handbook (which scales to input volume) using the following modifications:

·         PBS is used to normalize input volume between plasma samples

·         Do not use the provided carrier RNA which is typically dissolved in the AVE buffer

·         After adding the ACL buffer, incubate at 60°C for 45 minutes not 30 minutes as instructed

·         Elute from column using 30 µL nuclease free water. After elution, use 26 µL in the Dnase treatment

Dnase treatment and RNA Cleanup XP purification

1)      Add 3 µL of 10X Turbo buffer to 26 µL of sample

2)      Add 1 µL of Turbo Dnase

3)      Incubate at 37°C for 20min

4)      During incubation bring AMPure XP beads to room temp

5)      Once incubation is complete add 60 µL of AMPure XP beads to Dnase treated sample

6)      Incubate for 10 minutes

7)      Wash 2X with 80% ethanol

8)      Spin at 280 x g for 5 seconds

9)      Remove any residual ethanol with p20 pipette – Do not dry beads

10)   Elute in 9 µL of nuclease free water and transfer 8.5µL into a new well. Do not freeze sample and proceed to cDNA synthesis using the TruSeq RNA Exome Kit.

CDNA synthesis, library prep and exome enrichment using the TruSeq RNA Exome kit

Use the instructions in the TruSeq RNA exome reference guide as written.

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