Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/09/16

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngcDNA prep for U-2 OS samples Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Overview

After harvesting U-2 OS samples over the past 2 weeks, I'll be purifying the RNA and converting into cDNA using the Superscript III first-strand synthesis kit (Life Technologies 18080-051).

Sample Overview

This is a time course experiment, with each time point containing two biological replicates and a control.

  • U-2 OS 24hr, replicate A
  • U-2 OS 24hr, replicate B
  • U-2 OS 24hr, negative control
  • U-2 OS 48hr, replicate A
  • U-2 OS 48hr, replicate B
  • U-2 OS 48hr, negative control
  • U-2 OS 72hr, replicate A
  • U-2 OS 72hr, replicate B
  • U-2 OS 72hr, negative control

Samples were first grown to confluency in 6-well plates, then treated with 4 µg of KAH126-MV2 plasmid and transfected following the Lipofectamine LTX protocol. After incubation for the reported time, samples were harvested, measured via flow cytometry and fluorescence microscopy, and then digested in TRIzol before being stored at -80 °C.

RNA Concentration

Samples in TRIzol were treated using the TRIzol/RNeasy protocol. Samples were then measured using the BioTek plate reader.

Sample ID 260 280 260/280 ng/µL Vol. DNA (µL) Vol. water (µL)
U-2 OS 24hr A 0.254 0.123 2.058 203.277 24 (4.88 µg) 0
U-2 OS 24hr B 0.245 0.12 2.039 196.252 24 (4.71 µg) 0
U-2 OS 24hr neg 0.581 0.277 2.099 464.95 12.9 (6 µg) 11.1
U-2 OS 48hr A 0.322 0.158 2.033 257.467 23.3 (6 µg) 0.7
U-2 OS 48hr B 0.237 0.119 1.984 189.545 24 (4.54 µg) 0
U-2 OS 48hr neg 0.919 0.436 2.104 734.825 8.2 (6 µg) 15.8
U-2 OS 72hr A 0.78 0.371 2.102 624.205 9.6 (6 µg) 14.4
U-2 OS 72hr B 0.847 0.402 2.109 677.886 8.9 (6 µg) 15.1
U-2 OS 72hr neg 1.087 0.519 2.095 869.343 6.9 (6 µg) 17.1

Measured concentrations match what's expected from the treatment schedule - less RNA will be present in samples that were treated with plasmid due to cell death and inhibited growth compared to negative controls.

cDNA Synthesis

Running 3 reactions (6 µg) per sample, for a total of 27 reactions. Some samples have too low of an RNA concentration to load 2 µg per reaction, so I'll be running the max 8 µL instead.

oligo(dT) Primer-RNA annealing reactions

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50 μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
Water (SS III kit) = 8.0 - vol. total RNA
  10.0 μL
  • NOTE* To save on pipetting, I'll be setting up the above reactions with 30 µL total volume so there's 1 tube for each sample. Multiply all values by 3.

--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.


cDNA synthesis mix

  • Total reactions = 27

Samples (in triplicate):

  1. U-2 OS 24hr A
  2. U-2 OS 24hr B
  3. U-2 OS 24hr neg
  4. U-2 OS 48hr A
  5. U-2 OS 48hr B
  6. U-2 OS 48hr neg
  7. U-2 OS 72hr A
  8. U-2 OS 72hr B
  9. U-2 OS 72hr neg
Reagent Single rxn. Mix (x27)
10x RT buffer 2.0 54.0
25 mM MgCl2 4.0 108.0
0.1 M DDT 2.0 54.0
RNaseOUT 1.0 27.0
SuperScript III RT 1.0 27.0
  10.0 μL 270.0 μL

--> Aliquot 10 μL of mix into 8-tube strip
--> Add annealing rxn. into each 10 μL aliquot
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., 4°C/ ∞
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.
--> Store at -20°C