Quantitative / Real Time PCR

How does the rtPCR contract research work?

We discuss your specific requirements, after which we would need you to provide:

  • Assay information for your project; either with the sequence for the area, or, specific Applied Biosystems assays.
  • Enough total RNA to complete your work - this will be QC'd and converted into cDNA prior to running your assays on them to your specifications.

 

Results will be supplied in Excel in a mutually agreed format.

 

What QCs are carried out on the samples?

RNA samples are quality assessed using UV readings and the Agilent Bioanalyser. This gives an indication of the quality, and the amount of material present. Ensuring that material being converted into cDNA is of an acceptable quality.

 

What technology do you use for rtPCR?

We use the 5' Endonuclease assay (Taqman) to carry out real time reactions. This is a probe based technology where the assay is normally designed over an Exon / Exon boundary, this increases the specificity of the assay compared with sybr green based assays.

 

What is an endogenous control / Housekeeping gene - and do I need one?

When carrying out an rtPCR expression study it is very important to standardise data for variations in the extraction / conversion and running of the samples. The way this carried out is through the use of an endogenous or housekeeping gene - this will be a gene that has a fixed expression level that is unaffected by the experiment you are running. Without this control your data could be meaningless. Many researchers choose to run several control genes as an additional insurance.
Commonly used human control genes are:

  • Eukaryotic 18S rRNA
  • Human ACTB (beta actin)
  • Human B2M (beta-2-microglobulin)
  • Human GAPD (GAPDH)
  • Human GUSB (beta glucuronidase)
  • Human HPRT1 (HGPRT)
  • Human IPC (internal positive control)
  • Human PGK1 (phosphoglycerate kinase 1)
  • Human PPIA (cyclophilin A)
  • Human RPLP0 (large ribosomal protein)
  • Human TBP (TATA-box binding protein)
  • Human TFRC (CD71) (transferrin receptor)

 

How many replicate readings should I run?

We would recommend that you use three replicates for each sample. Duplicate readings are the bare minimum however occasionally larger deviations are seen - especially where very low expression levels are seen, triplicate readings makes it much more likely that usable data will be obtained throughout.

 

What size reaction volume do you use?

The reaction volume directly affects the cost of carrying out the work; we have found that 10ul reaction volumes generally give the most cost effective way of getting reproducible data. Lower reaction volumes can be run if requested, however we have found that results tend to have greater standard deviations.

 

Would the ABI Low-Density Array (LDA) be suitable for my project?

The Low-density array is a recent development from ABI in which assays from ABI's pre-validated list are spotted and dried onto a 384 array. From 11 to 380 assays can be arrayed in a limited number of arrangements. Each LDA has 8 ports each feeding 48 wells, into which your cDNA and master mix are added, once added the array is treated as a 384well plate and run on the ABI7900. The system benefits from low reaction volumes (1ul) and in our experience gives excellent reproducibility. However the main benefit is that it makes experiments with low numbers of samples, and larger numbers of assays more cost effective.

 

What equipment do you use?

Reactions are set up either manually using pipettes, or for larger projects a Matrix PlateMate Plus liquid handling robot is used. Plates are then heat sealed, as we have found that this greatly reduces the risk of evaporation and the associated reduction in data quality. The plates are run using an ABI 7900HT machine, and data analysed using ABI's SDS software.

 

What are the sample requirements?

Good quality total RNA should be supplied frozen and on dry ice. An electronic sample sheet should also be provided. Plates or tubes need to be clearly labelled. The amount of total RNA required is obviously dependent on the specific project, but for most projects 1.5-2?g of total RNA would be needed. RNA should be supplied in DEPC treated water at a minimum concentration of 100ng/ul.

 

How do I specify the assays I'm interested in running?

Assays can be specified in two ways:

  • 1: From ABI's list of over 600000 pre-validated assays from human, mouse, rat, Arabidopsis, and Drosophila genes. Assays can be searched for using ABI's search tool
    Please specify the assays using the ABI name, eg: Hs99999910_m1
  • 2: If an assay cannot found on ABI's list then we can get assays designed using the sequence of your target area. For this we need an assay name, and the sequence with the Exon / Exon boundary central and clearly marked. Ideally with at least 150bp on either side. From this sequence an assay will be designed with a primer in each exon and a probe spanning the Exon / Exon boundary.

 

How long will I have to wait to receive my data?

This obviously depends on the size of the project, and our current commitments. Generally however assays take in the region of 2 weeks to be delivered and the work is normally started soon after the assays arrive, most projects take 1-2 weeks to complete the wet work and return the data. An estimate of the project completion date will be given at the start of the work.

 

I still have some questions, who can I speak to?

Please contact us for further information. Our specialist will be more than happy to discuss your requirements in greater detail and can provide you with a custom proposal of work with pricing specific to your project.