User:Joytafty/Notebook/Effects of EphA2 and CXCR3 KD in hMVECs/2012/04/09

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RNA extraction of 2012/03/30 samples

Goal:

  • Assess the dynamics of commercialy (Qiagen) CXCR3 siRNA pool

Background:

  • hMVECs were transfected with Qiagen HiPerfect transfection reagents which were shown to efficiently deliver Cy3scrNeg at 4 h
  • hMVECs transfected with 75 nM EphA2 siRNA with 1:1.5 nM/µL RNAi-to-Qiagen HiPerfect ratio exhibits efficient EphA2 knockdown at longer time scale (48 - 120 h).

Materials:

  • 26g needles and syringes
  • RNAse free Eppendorb tubes
  • RNAse free pipette tips
  • Chloroform (Sigma, Calbiochem);
  • RNEasy Mini Kit (Qiagen 74104 (50) or 74106 (250);
  • DNAse/RNAse free water (MP Biomedicals 821739);
  • DEPC-treated water (Ambion AM9922, AM9915G);
  • RNase/DNase free tubes (VWR 87003-294);
  • 70 % Ethanol/RNAse free water

Procedure: Preparation

  • Spray down everything with RNaseAway water
  • Thaw sample on ice

Homogenize sample

  • Pass sample through 26g needle and syringe 12 - 15 times
  • Vortex to mix 5 - 10 s

Separate protein and DNA

  • Add 100 µL chloroform
  • Invert sample a few times by hand, then vortex 5 s
  • Let sit at RT 2-3 mins to allow sample to settle into 2 layers
  • COLD ROOM: Centrifuge 15 mins at 12000 RPM at 4C
  • warm DEPC water to 65 C, label Eppendorf collection tubes
  • Take sample from cold room centrifuge.
  • Carefully remove top layer of sample into labeled collection tubes
  • Keep track of sample volume (90 - 95 µL)
  • Put the bottom layer to -80C

KEEP ON ICE FROM THIS POINT ON

Collect RNA onto column

  • Add 150 µL 70% EtOH/RNaseAway water to sample. Mix by tipping/pipetting.
  • Remove sample and place in RNEasy column with collection tube.
  • Prepare RNAse free tubes to hold columns.
  • Centrifuge sample 15 s at > 10000 RPM
  • Return eluent to top of filter column
  • Repeat spin for 15 s at > 10000 RPM
  • Repeat a - c if sample remains

Buffer RW1 Rinse

  • Add 700 µL Buffer RW1 to columns
  • Centrifuge 15 s at > 10000 RPM
  • Discard flowthrough

RPE washes

  • Transfer sample to new collection tube
  • Add 500 µL RPE wash buffer to columns
  • Centrifuge 15 s at > 10000 RPM
  • discard flowthrough
  • Add another 500 µL RPE wash buffer
  • Centrifuge 2 min at > 10000 RPM
  • Transfer sample to new collection tubes
  • Centrifuge 1 min at > 10000 RPM to remove all ethanol

RNA elution

  • Add 20 µL DEPC water (heated to 65 C) to filter
  • Incubate filter at RT 2 - 3 mins
  • Centrifuge for 2 mins at > 10000 RPM in clean Eppendorf tubes to elute.

RNA purity check

  • Wipe everything down with RNAseAway water
  • Bring samples on ice to Nanodrop
  • Run samples on Nanodrop

How to run Nanodrop:

  • Open the Nanodrop Application. Select the “Nucleic Acid” tab.
  • Before being able to use the program, the Nanodrop needs to be initialized. Clean the sample pedestal RNAse free water and a Kimwipe. Add 2 uL of RNAse free water, put top arm down and click OK. Make sure that the liquid between the node surface of the top arm gets into contact with the pedestal. It should have an hourglass like appearance.
  • Select RNA-40 in the sample type window in the upper right corner.
  • Wipe off sample pedestal and blank the device with 2 uL of buffer that sample is in (from this protocol, it is DEPC-treated water).
  • Wipe off the pedestal and pipette 2 uL of the sample on to measure. Note: Read only a volume of sample once. If make an additional read on the sample, it will change. To get replicate readings, load another 2 uL of the sample on.

Result

Sample RNA Conc (ng/µL) A230/A280 A260/A280
24h1 75 nM CXCR3 RNAi Read1 value value value
24h1 75 nM CXCR3 RNAi Read2 value value value
24h1 50 nM CXCR3 RNAi Read1 value value value
24h1 50 nM CXCR3 RNAi Read2 value value value
24h1 25 nM CXCR3 RNAi Read1 value value value
24h1 25 nM CXCR3 RNAi Read2 value value value
24h1 10 nM CXCR3 RNAi Read1 value value value
24h1 10 nM CXCR3 RNAi Read2 value value value
24h1 5 nM CXCR3 RNAi Read1 value value value
24h1 5 nM CXCR3 RNAi Read2 value value value
24h1 0 nM CXCR3 RNAi Read1 value value value
24h1 0 nM CXCR3 RNAi Read2 value value value