CellSure cDNA Kit

Catalogue Numbers:


BIO-65040 30 Reactions
BIO-65041 100 Reactions

Features
  • Quick and easy production of cDNA directly from cell culture
  • No RNA extraction required
  • From cells to cDNA in 90 minutes

Applications
  • The cDNA is suitable for standard or Real-time PCR assays
  • High throughput gene expression analysis

Description


CellSure cDNA Kit is a convenient kit designed to quickly generate cDNA directly from cultured cells for analysis by PCR. The kit eliminates the need to purify RNA, which can be a time-consuming process and can lead to loss of sample, especially when starting material is limited. The kit is the ideal choice for researchers who wish to perform reverse transcription reactions on a small population of cells and provides sufficient cDNA for multiple PCR reactions.

A crude RNA extract is produced by a simple lysis step followed by heat treatment to inactivate RNases, and a DNase I treatment to degrade genomic DNA. The crude RNA extract is then used to synthesize cDNA using the reverse transcriptase provided. The kit can be used with a variety of mammalian cell lines including HeLa and NIH3T3.

CellSure cDNA Kit contains our reverse transcriptase, which is active over a wide range of temperatures.

Product Specifications


Batch details:
Batch No: See vial
Units per vial: See vial
Concentration: See vial

Storage Conditions:
CellSure cDNA kit contents can be stored for up to 6 months at -20°C except for the Mouse Total RNA, which should be stored at -80 °C.

Shipping Conditions:
On Dry Ice or Blue Ice

Associated Products:
 
Product Name Pack Size Cat No
IMMOLASE 250 Units BIO-21046
RiboSafe RNase Inhibitor 2500 Units BIO-65027
 

CellSure cDNA Kit Components
Component BIO-65040 30 Reactions BIO-65041 100 Reactions Description
Cell Lysis buffer 3ml 10ml Lyses the cells
1X PBS (pH 7.4) 30ml 100ml Removes serum proteins from the cells
DNase I (2u/μl) 60μl 200μl Degrades any DNA
5X Reverse Transcriptase Buffer 300μl 1ml Reverse Transcriptase Buffer
Reverse transcriptase 30μl 100μl Reverse Transcriptase
RNase inhibitor (10u/μl) 30μl 100μl High-affinity RNase inhibitor prevents degradation of RNA template
dNTP mix (10mM) 120μl 400μl High-purity (99%) dNTP mix manufactured by Bioline
Random Hexamers (50μM) 60μl 200μl 5’ NNNNNN 3’ for reverse transcription where transcripts are long or have significant secondary structure
Oligo (dT)18 (50μM) 60μl 200μl 5’ TTTTTTTTTTTTTTTTTT 3’ for reverse transcription where gene specific primers are designed close to the 3’
DEPC-H2O 1.75ml 2 x 1.75ml DEPC-treated H2O free of detectable RNase activity
Mouse Total RNA (1μg/μl) 10μl 10μl Control RNA
Control Primer mix (10μM) 10μl 10μl Control Primer mix
 

CellSure cDNA Kit Protocol

Overview:
A cell lysis buffer is used to lyse cells, which is followed by heat treatment to inactivate RNases. Contaminating genomic DNA is degraded by incubation with DNase I. A further heat treatment step inactivates the DNase I and the lysate is ready for reverse transcription.

Cell lysis and DNase I treatment:


The following protocol is for preparing a cell lysate with a 2 x 105 HeLa cells.
  • Count or estimate the number of cells.
    (i) Adherent cells: For cells grown in a 96-well plate, ensure that the final cell concentration does not reach >105 cells/well, since this would result in inhibition of the RT-PCR reaction. For cells grown in larger cell-culture vessels, detach the cells and then count the number.
    (ii) Suspension cells: Count cells directly in their growth medium.

  • Pipette 2 x 105 cells into a microcentrifuge tube. Centrifuge at 200x g for 5 minutes in a bench centrifuge to pellet the cells.
  • Remove the growth medium and wash cells at least once with 500μl of cold 1X PBS. For a 96 well plate, add cold 1X PBS directly to the cells in the well and discard.
  • Centrifuge as before, discard the supernatant and place the cells on ice.
  • Resuspend the cells in 100μl ice cold Cell Lysis Buffer. Control: For the control reaction, instead of the cells, add 1μl of Control Mouse Total RNA to 100μl of Cell lysis buffer. Cell lysate concentrations should be 1-2000 cells per μl of lysis buffer. To ensure optimum lysis conditions, do not lyse in more than 100μl, but do not exceed 2000 cells per μl as this may inhibit the RT-PCR reaction. For a small number of cells (≤10000 cells) or analysis of low-expressing genes, lyse the cells in a minimum of 5μl of lysis buffer. For single cell analysis, lyse in 5μl after which the whole lysate can be used for analysis by one-step RT-PCR.
  • Pipette up and down several times to mix and ensure cell disruption and leave on ice until the Cell Lysis Buffer has been added to all the samples. For a 96 well plate, lyse the cells directly in each well.
  • . Incubate at 75°C for 10 minutes to inactivate RNases and then place on ice to cool for 2 minutes.
  • Add DNase I to a final concentration of 0.04 U/μl and incubate at 37°C for 15 minutes to degrade genomic DNA.
  • Inactivate the DNase I by heating at 75°C for 5 minutes. 10. Place on ice and the lysate is ready for RT-PCR (lysate can be stored at -20°C at this stage).

Storage of the cell lysates:


Lysates made from ≥2.5 x 104 cells can be stored at -20°C for up to 1 week, or at -80°C for up to 2 months.
Lysates made from ≤2.5 x 104 cells should be used for RT-PCR immediately.

Two-step RT-PCR


Important Note: PCR components are not supplied with this kit.
No RT control: Include a No-RT control reaction with all components in the mix except for the reverse transcriptase.
  • For a 20μl reverse transcription reaction, assemble the following components in a microcentrifuge tube:
Component Amount Source
Cell lysate 5-10μl Generated by kit
Oligo dT and/or random hexamers * 2μl Supplied
dNTPs (10mM) 1μl Supplied
DEPC-treated H2O Up to 10μl Supplied
 
Either Oligo dT or random hexamers can be used to prime the RT reaction. Some users use a combination of Oligo dT and random hexamers in a molar ratio of 10:1 or 3:1 respectively, with a final concentration of 10μM per reaction. If a gene-specific primer is used to prime the RT reaction, its final concentration should be 0.25-5μM.
  • Heat at 70°C for 5 minutes.
  • Then add:
Component Amount Source
5x RT buffer 4μl Supplied
RNase Inhibitor 1μl Supplied
Reverse Transcriptase 0.25μl Supplied
DEPC-treated H2O Up to 20μl Supplied
 
  • Incubate at 42°C for 30-60 minutes.
  • Heat at 70°C for 10 minutes to inactivate the BioScript reverse transcriptase.
  • . Store the RT reaction at -20°C or proceed to the amplification step.
  • For a 50μl PCR reaction, assemble the following components on ice and mix by pipetting or gentle vortexing

    Control: For a control reaction, use the Control Primer mix with cDNA generated from the Control Mouse Total RNA lysate.
 
Component Amount Source
cDNA 1-5μl Generated by kit
10 x PCR buffer 5μl Not supplied
50mM MgCl2 1.5μl Not supplied
10mM dNTP mix 4μl Supplied
Forward primer 200-900 nM Control supplied
Reverse primer 200-900 nM Control supplied
Thermostable DNA polymerase 2 units Not supplied
Nuclease-free dH2O Up to 50μl Not supplied
Substitute forward and reverse primers with 1μl Control Primer mix in a 50μl PCR.
  • Program a thermal cycler for the following PCR conditions:
Temperature Duration Cycles
94°C 2 minutes 1
94°C 30 sec 30-40
Annealing temperature* 30 sec 30-40
72°C 30 sec 30-40
72°C 5 minutes minutes
1
 
* Start with an annealing temperature of 55°C
  • Analyse the amplified products

One-step RT-PCR


The lysate can also be used directly in a one-step RT-PCR reaction. We recommend 1-5μl of lysate in a 25μl reaction volume using the One-Step RT-PCR Kit (BIO-65030).

Quantitative PCR


The lysate is also suited to RT-PCR by real-time methods including two-step and one-step with both SYBR® Green and fluorescent probes.

General Considerations

Cell numbers
For different cell types, it may be necessary to optimise the number of cells for lysis, as there may be inhibitory factors present in your lysate that will inhibit the RT-PCR reaction.

Genomic DNA contamination


If genomic DNA contamination is still evident after DNase I treatment, use twice the amount of DNase I to provide a final concentration of 0.08U/μl in the lysate, or increase the incubation time to 30 minutes.

Primer Design


When designing primers it is important for RT-PCR that the primers flank at least one intron, or that one of the primers spans an exon-exon boundary in order to prevent any contaminating genomic DNA from being amplified. If the gene of interest has processed pseudogenes that may be present in the genomic DNA, then the PCR product from both the cDNA and the genomic DNA will be the same size. Wherever possible, design primers to avoid regions of secondary structure in the mRNA.

RNase Inactivation temperature


When inactivating RNases during the 10-minute incubation at 75°C, it is important to ensure that the temperature of the samples themselves reaches 75°C. To do so, use a calibrated heating device and heat well in advance. If necessary, check the temperature of a mock reaction using a thermometer with a microprobe.

CellSure cDNA Kit Troubleshooting Guide
Observation Possible Cause Recommended Solution(s)
No or low amounts of PCR products detected Insufficient PCR cycles Increase the number of cycles performed.
No or low amounts of PCR products detected RNases not completely inactivated Reduce the cell concentration in the cell lysis buffer. Ensure that in step 7 of the ‘Cell lysis and DNase I treatment’ section of the protocol, the cell lysate reaches 75°C.
No or low amounts of PCR products detected Cell lysate contains inhibitors of RT Reduce the number of cells added to the cell lysis buffer.
No or low amounts of PCR products detected RNA had high secondary structure Prior to reaction set-up, denature RNA with primers. Raise the temperature of the RT step, up to a maximum of 70°C (for short amplicons).
No or low amounts of PCR products detected RNA degradation To prevent RNA degradation, all buffers must be kept on ice.
Unspecific PCR products Non-specific annealing of primers to template Increase the annealing temperature.
Unspecific PCR products Primer dimers edesign primers to prevent self-annealing.
Product in no-RTase control Template contaminated with Genomic DNA Increase the incubation time of the DNase I treatment to 30 minutes. Increase the concentration of DNase I to 0.08U/μl.