Technical Bulletin

Cell Sample Quality

  • Important! – No QC will be performed for cultured cells prior to first-strand cDNA synthesis.  Please ensure you have selected cells with good viability.
  • We cannot work with cells that have undergone fixation.

Sample Amount Required

  • Our protocol is optimized for a starting input of 1 to 1000 cells.

General

  • cDNA synthesis from 1-1000 intact cells will be performed using SMART-Seq v4 Ultra Low Input RNA Kit (Clontech).
  • Due to the sensitivity of the protocol, the input material (cells) must be collected under clean-room conditions to avoid contamination.
  • Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (smaller tubes can crack when frozen).

Cell Preparation

  • It’s important that the cell culture medium does not inhibit first-strand cDNA synthesis.
  • Wash harvested cells 3 times with cell culture-grade PBS.
  • Bring the volume to 9.5 µl with nuclease-free water.
  • Add 1 µl of 10x Reaction Buffer (supplied by LC Sciences).  Gently vortex or pipette to mix the sample.
  • Important! – Immediately store the sample at -80 ºC and ship within 3 days on dry ice.
  • Indicate the number of cells in each sample on the sample submission form. The information is required for guiding the PCR cycle number in cDNA amplification.
  • International Orders – for those customers shipping samples oversees, please download a copy of our International Shipping Instructions.


Assessing heteroplasmic shifting induced by a therapeutic approach for the treatment of mitochondrial disease OligoMix® for Functional Analysis of Genetic Variants