Editing TREC Assay

== One Step DNA Extraction Method ==

DNA Elution from Dried Blood Samples Using a One-Step DNA Elution Buffer DNA Elution

1. Place 3.2 mm dried blood punches one per well into the wells of a multi-well plate.<br/>2. Add 54 μL of the one-step elution buffer into each well with an 8-Channel Pipette or an automatic liquid handler.<br/>3. Cover the plate with an adhesive cover.<br/>4. Centrifuge the plate at 3700 rpm for 5 minutes (optional).<br/>5. Heat the plate at 95° C. or greater for 25 minutes. Optionally, the eluted DNA solution is cooled at a temperature of at least about 4°C. for at least 5 minutes.<br/>6. Remove the plate from the thermal cycler, and allow the plate to cool to room temperature.

'''One step buffer'''<br/>15 mM Tris-Base<br/>10 mM KCl<br/>5 mM KOH, pH 11.5.







'''DNA Extraction from DBS-Multiple Steps Method'''<br/>A. Using an 8-Channel Pipette, add 90 μL of Qiagen® Gentra DNA Purification Solution (Solution 1) to each well containing a 3.2 mm dried blood disk.<br/>B. Centrifuge the plate at 3700 rpm for 30 seconds to ensure that the disks are immersed in the solution.<br/>C. Incubate the plate at room temperature for 15 minutes.<br/>D. Centrifuge the plate at 3700 rpm for 5 minutes.<br/>E. Using an 8-Channel Pipette, remove and discard as much of the liquid as possible into 10% bleach solution.<br/>F. Repeat step A-E, with Step C for 10 minutes.<br/>G. Using an 8-Channel Pipette, add 90 uL of autoclaved Milli-Q H2O to each well.<br/>H. Centrifuge the plate at 3700 rpm for 30 seconds.<br/>I. Using an 8-Channel Pipette, remove and discard as much liquid as possible into a 10% bleach solution.<br/>J. Using an 8-Channel Pipette, add 24 uL Qiagen® Gentra DNA Elution Solution (Solution 2) into each well.<br/>K. Centrifuge the plate at 3700 rpm for 30 seconds and then cover with adhesive cover.<br/>L. Place the plate in the −20° C. freezer for a minimum of 30 minutes.<br/>M. Remove the plate from the freezer and allow it to thaw.<br/>N. Centrifuge the plate. Allow the centrifuge to ramp up to 3700 rpm, wait 10-30 seconds, and stop the centrifuge. Remove the plate.<br/>O. Place the plate into Veriti™ 96-Well Thermal Cycler.<br/>P. Heat the plate at 99° C. for 30 minutes.<br/>Q. After 99° C. incubation, remove the plate from the thermal cycler and allow the plate to cool to room temperature, remove the plate from the thermal cycler, and allow the plate to cool to room temperature.<br/>R. Centrifuge the plate at 3700 rpm for 30 seconds.

<span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 13.3333px;">Serially diluted plasmids containing known numbers of TRECs can be used to produce a calibration curve. A TREC cut-off level of 75 or less TRECs per 3.2 mm dried blood sample is used to identify newborns with SCID. The primers and probes used in the SCID assay are in Table 6. A β-actin primer pair serves as an optional control for DNA elution from the dried blood spots.</span>

Primer name&nbsp;&nbsp; &nbsp;Sequence&nbsp;<br/>TREC forward primer&nbsp;&nbsp; &nbsp;5′-CACATCCCTTTCAACCATGCT-3′&nbsp;<br/>TREC reverse primer&nbsp;&nbsp; &nbsp;5′-GCCAGCTGCAGGGTTTAGG-3′&nbsp;<br/>β-actin forward&nbsp;&nbsp; &nbsp; primer 5′-ATTTCCCTCTCAGGAATGGA-3′&nbsp;<br/>&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;<br/>β-actin reverse primer&nbsp;&nbsp; &nbsp;5′-CGTCACACTTCATGATGGAGTTG-3 ‘<br/>TREC PROBE&nbsp;&nbsp; &nbsp;6-FMA-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-3′TAMRA&nbsp;&nbsp; &nbsp;<br/>β-actin probe&nbsp;&nbsp; &nbsp;6-VIC-GTGGCATCCACGAAACTA-3′-TAMRA&nbsp;

Reagents per reaction&nbsp;&nbsp; &nbsp;μL<br/>Gene Expression Master Mix&nbsp;&nbsp; &nbsp;10<br/>TREC forward primer (10 uM)&nbsp;&nbsp; &nbsp;1<br/>TREC reverse primer (10 uM)&nbsp;&nbsp; &nbsp;1<br/>Probe (15 uM)&nbsp;&nbsp; &nbsp;0.2<br/>BSA (10 mg/ml)&nbsp;&nbsp; &nbsp;0.8<br/>DNA—from an eluted DNA&nbsp;&nbsp; &nbsp;6

1. 50° C. for 2 min.<br/>2. 95° C. for 10 min.<br/>3. 95° C. for 30 sec.<br/>4. 62° C. for 60 sec.<br/>5. Repeat 3-4 for 40 cycles<br/>6. 4° C. forever

<br/>For data collection, a fixed cycle threshold (Ct) was set at the point when PCR amplification is still in the exponential phase. The cycle at which the instrument can discriminate the amplification generated fluorescence from the background noise is called the threshold cycle (Ct). FIG. 3 shows the successful RT-qPCR amplification plots for Quantification of T-cell Receptor Excision Circles (TREC) assay on DNA samples extracted by using the method described here. The number of TRECs are automatically determined based on serially diluted plasmids containing a known copy number of TRECs



Translational Centre for Regenerative Medicine, Leipzig, Germany: pre-prepared PCR master mix, standard samples and control samples

PCR master mix contained specific oligonucleotides and hydrolysed probes for TREC, KREC and ACTB specific detection.

TREC probes were labelled with FAM fluorochrome, KREC probes labelled with VIC fluorochrome and ACTB probes labelled with NED fluorochrome.

The master mix also contained MgCl2, bovine serum albumin (BSA), ROX dye, E.coli uracil-DNA glycosylase (UDG) and specific dUTP:dTTP nucleotide ratio.

Five standard samples were analysed in order to make a standard curve. The standard samples were in a tenfold dilution, from 100.000 to 10 copies/μL for TREC and KREC and from 1.000.000 to 100 copies/μL for ACTB. The two least concentrated standard samples were analysed in triplicates and higher standard samples in duplicates. Five control samples were analysed in each RT-qPCR run, normal control (NC), T cell depleted sample with no TREC (TCD), B cell depleted sample with no KREC (BCD), T and B cell depleted sample with neither TREC nor KREC (TBCD) and no template control sample with no DNA present (NTC-PCR).

12 μL of the PCR master mix and 8 μL of sample were added to each well of a white 384 well PCR plate (Thermo scientific, MA, USA) with low retention/binding SafeSeal Tips (Biozym scientific GmbH, Hess. Oldendorf, Germany). Plates were sealed with Optical adhesion covers (Applied Biosystems® before inserted into the ABI PRISM® 7900HT (Applied Biosystems®).

The thermal cycle program is shown in Table 1. The first step was UDG activation at 50°C for two minutes. The second step was DNA denaturation and activation of the polymerase at 95°C for ten minutes, followed by 40 cycles of amplification at 95°C for 15 seconds and fluorescence detection at 60°C for 25 seconds. Ramp rate was set to 100% or 1,6 °C/sec.

Sensitivity, specificity, positive and negative likelihood ratio and positive and negative predictive values (P/NPV) for both TREC and KREC RT-qPCR method were calculated using the equations 2─5. Inconclusive results (ACTB <1000 copies/μL) and repeated measurements were excluded from the calculations.

Another Protocol Briefly, each PCR reaction was conducted in a 20 mL mixture containing 12.5 mL of 26 Taqman Mastermix, 1.25 mL each of forward primer, reverse primer and probe (containing a quencher and reporter dye sybr green) and 3.75 mL of water. The sequences of the primers and probe used previously [31] are: forward primer 59-CCATGCTGACACCTCTGGTT-39, reverse primer 59-TCGTGAGAACGGTGAATGAAG-39 and the probe 59- CACGGTGATGCATAGGCACCTGC-39. To nomalise for the amount of DNA the Ca constant region of the TCR was also amplified using the following: forward primer 59- CCTGATCCTCCTGTCCCACAG-39, reverse primer 59- GGATTTAGAGTCTCTCAGCTGGTACA-39, and the probe 59-ATCCAGAACCCTGACCCTGACCCTGCCG-39. The PCR conditions were: 50uC for 2 min; 95uC for 10 min; 50 cycles of amplification (95uC for 15 s; 60uC for 1 min). All samples were run in duplicate on a Rotogene PCR machine

Copy number for the δ Rec-ψJα sj TREC and a β-Actin gene segment as a control were determined for duplicate 5 uL samples of the above DNAs using the ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA). Standard curves were generated from serial dilutions of TREC and β-Actin plasmids. PCR reactions (20 uL) contained BSA 0.04% (New England Biolabs, Ipswich, MA), 1X TaqMan Universal Master mix, 150 nmol/L of FAM-TAMRA fluorescent probe and 500 nmol/L of forward and reverse primers (Applied Biosystems, Foster City, CA) [12,13]. After 2 min at 50°C and 10 min at 95°C, 40 cycles were conducted, 30 s at 95°C and 1 min at 60°C. Comparison copy number values for normal infants per 3.2mm punch were TREC’s >100, β-Actin >100,000, based on 2000 anonymous newborn DBS samples from the California Department of Public Health [J. Puck and D. Gonzalez, unpublished data]. Samples from T cell deficient patients [9, and unpublished], have <100 TRECs per punch. If β-Actin copies are <100,000, the sample is considered not adequate due to amplification failure.
[[Category:QPCR RT-PCR]] [[Category:Protocols]]