4.10.1. External reference standards

The classic way to relate indirect measurements to absolute amounts of target is through external reference standards. These are established by running the RT-qPCR assay on a dilution series of a known amount of target (external standard), using the resulting data to calculate the relationship between the absolute amount of target and cycle number, and then using this equation to convert the sample data to absolute amounts (Bustin, 2000). All modern real-time PCR thermocyclers have this function automatically included in their software, requiring as only input the absolute concentrations of the external standards. Such curves are also extremely useful during optimization of the RT-qPCR reaction conditions, particularly for determining the reaction efficiency (Bustin et al., 2009).

External reference dilution standards should be prepared for all targets, including the internal reference standards. This is done, in short, by amplifying the appropriate fragment with PCR, purifying and cloning this fragment in a plasmid and preparing purified, well quantified plasmid DNA. This plasmid DNA can be either used directly to prepare DNA-based external standard series, or be used to synthesize RNA transcripts which in turn are quantified accurately and used to prepare RNA-based external standard series. DNA-based standards tend to be more sensitive and reproducible but RNA-based standards are more realistic and also take the cDNA reaction efficiency into account. The professional literature is divided on the issue, with good arguments for both approaches (Pfaffl and Hageleit, 2001). Both curves still require several positive control RNA samples per run, to normalize between runs for differences in reagent mixtures and, in the case of the DNA curve, to account for the reverse transcription step as well.

Reference standards from PCR products:

  1. Amplify the target fragment by RT-PCR.
  2. Confirm the amplification and absence of secondary products with electrophoresis.
  3. If there are secondary products, excise the correct fragment under low-intensity UV light.
  4. Purify the fragment using a commercial DNA


The purified PCR fragments can be used directly to prepare an external reference standard, as follows:

  1. Estimate the DNA concentration of the fragment in ng/µl, using spectrophotometry (e.g. Nanodrop, section 3.2.1) or fluorimetry (e.g. Qubit®; www.inVitrogen.com).
  2. Estimate the molecular weight of your fragment.
    This can be done exactly, based either on actual sequence or on fragment length, in the tools tab at www.currentprotocols.com. An approximate estimate for fragments within the 100-1000 bp range is:
    MWdsDNA = bp x 617 ng/nmol
  3. Convert the DNA concentration to copies/µl as follows:
    copies/µl = [ng/µl]/[MWdsDNA] x [6.0221415 x 1014 copies/nmol]
  4. Store the undiluted DNA fragment in aliquots at -80oC.
  5. Prepare a working quantification standards series by serial ten-fold dilution of the DNA fragment, ranging from 1012 – 100 copies/µl, in 10 ng/µl yeast or E. coli tRNA (Bustin et al., 2009), to minimize loss of standard DNA due to adsorption to the microcentrifuge tube walls.

 

Whether or not the PCR products are used directly for preparing external reference standards, they should also be cloned: for confirmation of the fragment by sequencing, for long-term preservation of a positive DNA control and for the synthesis of RNA-based external reference standards. The fragment should be cloned into a T/A plasmid cloning vector that has T7 and T3 RNA promoters either side of the cloning site. Many molecular supply companies market such T/A cloning vectors, which are specially prepared for cloning PCR fragments.

  1. Clone PCR fragments.
    Protocols for cloning PCR fragments are beyond the scope of this paper. For this, the reader is referred to the product manuals provided by commercial suppliers of T/A cloning kits, and specialist manuals, such as the outstanding and long-established “Molecular cloning: a laboratory manual“, by Green and Sambrook (2012).
  2. Confirm candidate bacterial clones by colony PCR. This is a conventional 20 µl PCR reaction using the primers and amplification profile appropriate for the target, containing a small smudge of primary bacterial colony as template. 
  3. Run the colony-PCR products on an agarose gel (Green and Sambrook, 2012, see section 3.2.1).
  4. Identify those colonies containing a plasmid with a cloned target.
  5. Prepare small-scale liquid cultures of positive bacterial clones (Green and Sambrook, 2012).
  6. Mix 0.5 ml of liquid bacterial culture with 0.5 ml 50% sterile glycerol and store this at -20oC (glycerol stocks).
  7. Prepare plasmid DNA from the remaining liquid bacterial culture, using either a commercial plasmid purification kit or home-made reagents recommended in a molecular laboratory manual (Green and Sambrook, 2012).
    Make sure that the protocol includes an RNAse step, to digest any bacterial RNA.
  8. Purify the plasmid DNA on a commercial DNA affinity purification column.
  9. Sequence the plasmid, using universal plasmid-based primers.
    This is best done at specialist commercial facilities.
  10. Confirm the presence of the insert in the plasmid from the sequence data, and the orientation of the insert in the plasmid.
  11. Estimate the DNA concentration of the plasmid in ng/µl, using spectrophotometry (e.g. Nanodrop, section 3.2.1) or fluorimetry (e.g. Qubit®; www.InVitrogen.com).
    dsDNA             A260 1,0 = 50 ng/µl
  12. Estimate the molecular weight of the plasmid + insert, by combining their lengths in bp and converting either exactly at www.currentprotocols.com or approximately as follows:
    MWdsDNA = (bpplasmid + bpinsert) x 607.4 + 157.9 ng/nmol
  13. Convert the DNA concentration to copies/ul as follows:
    copies/µl = [ng/µl]/[MWdsDNA] x [6.0221415 x 1014 copies/nmol]
  14. Store the undiluted plasmid in aliquots at -80oC.
  15. Prepare a working quantification standards series by serial ten-fold dilution of the plasmid, ranging from 1012 – 100 copies/µl, in 10 ng/µl yeast or E. coli tRNA (Bustin et al., 2009).

 

RNA-based external reference standards

  1. Transcribe RNA from purified plasmid DNA, using either the T7 or the T3 promoter, depending on the orientation of the insert and the desired strand polarity of the RNA.
  2. Linearize the plasmid with a restriction enzyme that digests right after the cloned fragment, in the desired orientation.
    This ensures that the RNA transcripts have a defined length.
  3. Transcribe the digested plasmid with a specific commercial T3/T7 RNA transcription kit.
    Follow the corresponding instructions. Alternatively, detailed protocols with home-made reagents can be found in Green and Sambrook (2012).
  4. Digest the synthetic, transcribed RNA with DNAse, as recommended by the kit manufacturer.
    This is to remove contaminating plasmid DNA which may co-amplify and thus interfere with correct quantification.
  5. Purify the DNAse-treated RNA on RNA affinity purification columns.
  6. Estimate the RNA concentration in ng/µl, using spectrophotometry (e.g. Nanodrop, section 3.2.1) or fluorimetry (e.g. Qubit®; InVitrogen).
    ssRNA           A260 1,0 = 40 ng/µl
  7. Calculate the insert size (number of bases from the T3/T7 promoter site to the restriction enzyme site on the other side of the insert used for digesting the plasmid).
  8. Estimate the molecular weight of the RNA transcript either exactly at www.currentprotocols.com or approximately as follows:
    MWssRNA = nt x 320.5 + 159.0 ng/nmol
  9. Convert the concentration of the synthetic RNA to copies/µl as follows:
    copies/µl = [ng/µl]/[MWssRNA] x [6.0221415 x 1014 copies/nmol]
  10. Store the undiluted RNA in aliquots at -80oC.
  11. Prepare a working quantification standards series by serial ten-fold dilution of the RNA, ranging from 1012 – 100 copies/µl.

Do not use an RNA carrier for preparing the dilution series, since this carrier RNA will participate in the reverse transcriptase reaction and thereby significantly affect quantification!! Instead, dilute either in nuclease-free water or in 10 ng/µl of a neutral DNA carrier, obtained from a commercial source.

 

 

The BEEBOOK