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
Results will be supplied in Excel in a mutually agreed
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
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
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:
- 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?
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