Editing TREC ASSAY (section)

== Standard Curve Preparation ==

Clone # 2489 pMiniT-TREC-ACTB-ALB



Clone # 2489 pMiniT-TREC-ACTB-ALB

Plasmid Data:

Length: 3515 bp,

MW: 1085647.7, 50% GC

OD=1: 27nM, 29.3 ug

EXTINCTION COEFFICIENT 33521600 L/(mole·cm)

nmole/OD260:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.027

µg/OD260:&nbsp;&nbsp; 29.3

1085647.7 g = 1 mole=6.023 * 10<sup>23</sup> molecules (copies)

1 mmole= 6.023 * 10<sup>20</sup> copies

1 umole= 6.023 * 10<sup>17</sup> copies

1 nmole= 6.023 * 10<sup>14</sup> copies

&nbsp;

Mass of one plasmid molecule= 3515 X bp 1.096<sup>-21</sup> g = 3.85<sup>-18</sup> g

100,000 copies of beta actin or TREC:

3.85-18 g X 100,000 copies = 3.85<sup>-13</sup> g

the average mass per bp is 1.096 x 10<sup>-21</sup> g/bp, and therefore:

mp = (3515 bp) x (1.096 x 10<sup>-21</sup> g/bp) = 3852.44 x 10<sup>-21</sup> g = 3.852 x 10<sup>-18</sup> g.





Table 1

{| border="1" cellpadding="1" cellspacing="1"
|-
| Copy #
| rowspan="6" | x 3.852 x 10<sup>-18</sup> g
| Mass of plasmid DNA (g)
| <br/>
|-
| 1 x 10<sup>6</sup>
| 3.852 x 10<sup>-12</sup>
| 3.852 pg&nbsp;<br/>
|-
| 1 x 10<sup>5</sup>
| 3.852 x 10<sup>-13</sup>
| <br/>
|-
| 1 x 10<sup>4</sup>
| 3.852 x 10<sup>-14</sup>
| <br/>
|-
| 1 x 10<sup>3</sup>
| 3.852 x 10<sup>-15</sup>
| <br/>
|-
| 1 x 10<sup>2</sup>
| 3.852 x 10<sup>-16</sup>
| <br/>
|}

'''Calculation'''

NOTE: Prepare all dilutions in 0.1 x TE buffer in a place specially designed for containment of DNA carry-over.

Calculate the mass of the plasmid copy numbers of interest (see '''Table 2'''), taking into account that the plasmid size is 3515 bp and that the average mass per bp is 1.096 x 10<sup>-21</sup>g/bp, and therefore: mp= (3515 bp) x (1.096 x 10<sup>-21</sup>g/bp) = 3852.44 x 10<sup>-21</sup>g = 3.852 x 10<sup>-18</sup>g.

'''Table 2. Mass of plasmid needed for each standard curve dilution point.'''

Calculate the concentrations of plasmid DNA by dividing the needed plasmid masses by the volume (5 μl) to be pipetted into each reaction (see '''Table 3''').



{| border="1" cellpadding="1" cellspacing="1"
|-
| Copies
| Mass of plasmid DNA (g)
| &nbsp;
| Final concentration of plasmid DNA (g/μl)<br/>
|-
| 1 x 10<sup>6</sup>
| 3.852 x 10<sup>-12</sup>
| &nbsp;
| 7.704&nbsp;x 10<sup>-13</sup>
|-
| 1 x 10<sup>5</sup>
| 3.852 x 10<sup>-13</sup>
| &nbsp;
| 7.704 x 10<sup>-14</sup>
|-
| 1 x 10<sup>4</sup>
| 3.852 x 10<sup>-14</sup>
| ÷ 5 μl
| 7.704 x 10<sup>-15</sup>
|-
| 1 x 10<sup>3</sup>
| 3.852 x 10<sup>-15</sup>
| <br/>
| 7.704 x 10<sup>-16</sup>
|-
| 1 x 10<sup>2</sup>
| 3.852 x 10<sup>-16</sup>
| <br/>
| 7.704 x 10<sup>-17</sup>
|}

'''Table 3. Calculation of plasmid concentrations needed for each dilution point.'''

'''Dilution'''

Thaw an aliquot of the plasmid.

Linearize 2 μg of the plasmid DNA with EcoRV&nbsp;and determine its concentration by spectrophotometric estimation at 260/280 nm.

Prepare a proper dilution of the linearized plasmid DNA in order to achieve a convenient working stock solution to start from (''e.g.,'' 100 ng/μl = 0.1 μg/μl = 1 x 10<sup>-7 </sup>g/μl).

Store the remainder of the linearized plasmid DNA at -80 °C.

Perform a convenient number of 1:10 or 1:100 dilutions to bring the plasmid at the more workable concentration of 1 x 10<sup>-10</sup> g/μl.

Use the formula C<sub>1</sub>V<sub>1 </sub>= C<sub>2</sub>V<sub>2 </sub>to calculate the dilution volume (V<sub>2</sub> – V<sub>1</sub>) needed to prepare the first standard curve point of the series (1 x 10<sup>6</sup> copies, corresponding to 7.704 x 10<sup>-13</sup>&nbsp;g/5 μl; see '''Table 4''').

To make 100 ng /ul from 1360 ug/ul: 73.5 ul of 1360 ng/ul plasmid stock, + 926 ul of TE.



{| border="1" cellpadding="1" cellspacing="1"
|-
| <br/>
| Name<br/>
| Initial conc. (g/μl)&nbsp;
| Plasmid DNA vol (μl)&nbsp;
| Diluent vol (μl) TE&nbsp;
| Final Vol. (μl)&nbsp;
| Final conc. (g/μl)&nbsp;
| rowspan="2" | Final copy number of plasmid DNA/5μl&nbsp;
| rowspan="2" | <br/>
|-
| #
| <br/>
| C<sub>1</sub>
| V<sub>1</sub>
| V<sub>2</sub>-V<sub>1</sub>
| V<sub>2</sub>
| C<sub>2</sub>
|-
| #1
| MaxiPrep Stock<br/>
| &nbsp;1360 ng/μl
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| #2
| 100 ng/μl
| 1360 ng/μl<br/>
| 50&nbsp;μl
| 630
| 680
| 100 ng/μl<br/>
| <br/>
| <br/>
|-
| #3&nbsp;
| 10 ng/μl
| 1 x 10<sup>-7&nbsp;</sup><span style="font-size: 10.8333px;">(100ng/</span>μl<span style="font-size: 10.8333px;">)</span>
| 5 μl
| 45 μl
| 50 μl
| 1 x 10<sup>-8</sup>&nbsp;,10ng/μl
| N/A
| <br/>
|-
| #4&nbsp;
| 100 pg/μl
| 1 x 10<sup>-8 </sup>(10 ng/μl)
| 5 μl
| 495 μl
| 500 μl
| 1 x 10<sup>-10 </sup>,100 pg/μl
| N/A
| <br/>
|-
| #5&nbsp;
| 10<sup>6</sup>/5μl
| 1 x 10<sup>-10</sup> (100pg/μl<sup>)</sup>
| 5 μl
| 644 μl
| 649 μl
| 7.704 x 10<sup>-13</sup>
| 1 x 10<sup>6</sup>
| <br/>
|-
| #6&nbsp;
| 10<sup>5</sup>/5μll<br/>
| 7.704&nbsp;&nbsp; x 10<sup>-13&nbsp;</sup>(10pg/μl<sup>)</sup>l)
| 5 μl
| 45 μl
| 50 μl
| 7.704 x 10<sup>-14</sup>
| 1 x 10<sup>5</sup>
| <br/>
|-
| #7&nbsp;
| 10<sup>4</sup>/5μl<br/>
| 7.704&nbsp;&nbsp; x 10<sup>-14 </sup>(1pg/μl)
| 5 μl
| 45 μl
| 50 μl
| 7.704 x 10<sup>-15</sup>
| 1 x 10<sup>4</sup>
| <br/>
|-
| #8&nbsp;
| 10<sup>3</sup>/5μl<br/>
| 7.704&nbsp;&nbsp; x 10<sup>-15&nbsp;</sup>(100fg/μl)
| 5 μl
| 45 μl
| 50 μl
| 7.704 x 10<sup>-16</sup>
| 1 x 10<sup>3</sup>
| <br/>
|-
| #9&nbsp;
| 10<sup>2</sup>/5μl<br/>
| 7.704 &nbsp; x 10<sup>-16</sup>(10fg/μl)
| 5 μl
| 45 μl
| 50 μl
| 7.704 x 10<sup>-17</sup>
| 1 x 10<sup>2</sup>
| <br/>
|-
| #10
| 10/5μl
| 7.704 &nbsp; x 10<sup>-17</sup>(1fg/μl)<br/>
| 5 μl<br/>
| 45 μl<br/>
| 50 μl<br/>
| 7.704 x 10<sup>-18</sup><br/>
| 10 prepare fresh
| <br/>
|}

'''Table 4. Dilution calculations.'''

Proceed with a regular series of 1:10 dilutions to obtain the remaining standard curve points.<br/>NOTE: Do not forget to vortex each plasmid dilution briefly before performing the next.<br/>NOTE: The highest dilution of the standard curve (10 copies/5 μl) must be prepared just before performing the assay, because such a small quantity of plasmid DNA is not stable enough to be stored for future use.

'''DNA Extraction from Target Samples'''

#Separate PBMC from peripheral blood collected into EDTA tubes using the density gradient separation method.<br/>NOTE: Alternatively, use other appropriate starting material (''e.g.,'' sorted lymphocyte subpopulations, whole blood) as target sample.
#Extract the DNA from PBMC of target samples

DNA Extraction from DBS / 96 well format

<br/>1. A 3 mm punch is made on the specimen card and the punched disk is collected into 96 well Reaction Plate *&nbsp;<br/>2. Make 5 empty punches on a paper towel to avoid cross contamination between samples<br/>3. Add 90 ul Solution 1* to each well. Close the plate with Clear Adhesive Film *<br/>4. Spin the plate at 3700 rpm 1' to immerse the disks<br/>5. Leave the plate at RT for 15'<br/>6. Spin at 3700 rpm 5'<br/>7. Using a pipette, remove and discard (safely) the liquid<br/>8. Repeat steps 3-7<br/>9. Add 90 ul Solution 2*<br/>10. Spin at 3700 rpm 5'<br/>11. Using a pipette, remove and discard the liquid<br/>12. Add 24 ul Nuclease-Free Water<br/>13. Place the plate at -20 deg C for 30' to overnight (overnight works better)<br/>14. Take the plate out and let it warm to room temperature<br/>15. Spin at 3700 rpm 1'<br/>16. Place the plate in a thermal cycler for 25' 99 deg C<br/>17. Remove the plate and let it cool down to room temperature<br/>18. Spin 3700 rpm 1'&nbsp;<br/>19. Store the DNA Extracts at -20 deg C until use<br/>15. Use 4 ul (mix / pipette gently before taking an aliquot) of the DNA Extract to assay for the TREC

A 3.2-mm disk of DBS from each NBS card was punched into a MicroAmp Optical 96-Well Reaction Plate well&nbsp;(Applied Biosystems, Foster City, Calif) using a standard MultiPuncher (PerkinElmer, Waltham, Mass) and washed twice with 90 uL Purification Solution, followed by washing once with 90 uL of Elution Solution. A total of 24 uL autoclaved sterile water was then added to each well, and the plate was chilled in a –20 oC freezer for a minimum 15 minutes before being heated at 99 oC for 25 minutes. To assess whether cross-contamination occurred among the different wells in a 96-well plate, a plain filter paper disk was&nbsp;placed into a well after every seventh DBS. TRECs were not detected in any wells containing a blank filter paper disk, indicating a lack of sample cross-contamination&nbsp;



DBS Sampling &nbsp;<br/>DBS sampling is popular and advantageous because of many factors: ease of obtaining sample by finger / heel stick, ease of transport and sample stability at ambient temperature or under refrigeration for many years.<br/>About 10-20 ul of whole blood is spotted on a DBS card or approved filter paper cards (Whatman 903 filter paper). The spots are allowed to dry for 24 hours and then stored at 4 deg C (-20 deg C for long term storage) or processed.<br/>* Reagents and Supplies for DNA Extraction from DBS &nbsp;<br/>MicroAmp Optical 96-Well Reaction Plate (ABI)<br/>MicroAmp Clear Adhesive Film (ABI)<br/>Solution 1 = Generation DNA Purification Solution (QIAGEN)<br/>Solution 2 = Generation DNA Elution Solution (QIAGEN)



DBS Sampling DBS sampling is popular and advantageous because of many factors: ease of obtaining sample by finger / heel stick, ease of transport and sample stability at ambient temperature or under refrigeration for many years. About 10-20 ul of whole blood is spotted on a DBS card or approved filter paper cards (Whatman 903 filter paper). The spots are allowed to dry for 24 hours and then stored at 4 deg C (-20 deg C for long term storage) or processed.

*Reagents and Supplies for DNA Extraction from DBS

MicroAmp Optical 96-Well Reaction Plate (ABI) MicroAmp Clear Adhesive Film (ABI) Solution 1 = Generation DNA Purification Solution (QIAGEN) Solution 2 = Generation DNA Elution Solution (QIAGEN)

'''&nbsp;Real-time PCR for Quantification of TRECs'''

#Plate preparation and loading:
##Arrange a PCR plate including at least two replicates for each samples to be analyzed: standard curve (A→F 1→4), positive control (CTRL<sup>+</sup>; G1→4) and no-template control (NTC; H 1→4).<br/>NOTE: Observe a typical scheme in '''Table 5''', but different formats can be created. Remember that TRECs and Beta Actin&nbsp;will be amplified together in the same wells.

{| border="0" cellpadding="0" cellspacing="0"
|-
| &nbsp;
| colspan="2" | TRECs+ACTB
| colspan="2" | ACTB
| colspan="2" | TRECs+KRECs
| colspan="2" | TCRAC
| colspan="2" | TRECs+KRECs
| colspan="2" | TCRAC
|-
| &nbsp;
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
|-
| A
| 10<sup>6</sup> copies
| 10<sup>6</sup> copies
| 10<sup>6</sup> copies
| 10<sup>6</sup> copies
| 1
| 1
| 1
| 1
| 9
| 9
| 9
| 9
|-
| B
| 10<sup>5</sup> copies
| 10<sup>5</sup> copies
| 10<sup>5</sup> copies
| 10<sup>5</sup> copies
| 2
| 2
| 2
| 2
| 10
| 10
| 10
| 10
|-
| C
| 10<sup>4</sup> copies
| 10<sup>4</sup> copies
| 10<sup>4</sup> copies
| 10<sup>4</sup> copies
| 3
| 3
| 3
| 3
| 11
| 11
| 11
| 11
|-
| D
| 10<sup>3</sup> copies
| 10<sup>3</sup> copies
| 10<sup>3</sup> copies
| 10<sup>3</sup> copies
| 4
| 4
| 4
| 4
| 12
| 12
| 12
| 12
|-
| E
| 10<sup>2</sup> copies
| 10<sup>2</sup> copies
| 10<sup>2</sup> copies
| 10<sup>2</sup> copies
| 5
| 5
| 5
| 5
| 13
| 13
| 13
| 13
|-
| F
| 10 copies
| 10 copies
| 10 copies
| 10 copies
| 6
| 6
| 6
| 6
| 14
| 14
| 14
| 14
|-
| G
| CTRL<sup>+</sup>
| CTRL<sup>+</sup>
| CTRL<sup>+</sup>
| CTRL<sup>+</sup>
| 7
| 7
| 7
| 7
| 15
| 15
| 15
| 15
|-
| H
| NTC
| NTC
| NTC
| NTC
| 8
| 8
| 8
| 8
| 16
| 16
| 16
| 16
|}

'''Table 5. Sample real-time PCR plate.'''

Calculate the total number of wells needed (include 1 - 2 extra wells to account for possible pipetting errors) and prepare the PCR master mix for TRECs/KRECs and TCRAC in separate tubes (see '''Table 6'''). &nbsp;&nbsp;NOTE: The primers and probes specific for TRECs, KRECs and TCRAC are mentioned in '''Table 7'''. The primer and probe final concentrations are 900 nM and 200 nM, respectively.

{| border="0" cellpadding="0" cellspacing="0"
|-
| colspan="2" | TRECs/KRECs
| colspan="2" | TCRAC
|-
| H<sub>2</sub>O
| 2 μl
| H<sub>2</sub>O
| 4.75 μl
|-
| ACTB F 10 pmol/μl
| 1&nbsp;μl
| TCRAC for 20 pmol/μl
| 1.125 μl
|-
| ACTB R 10 pmol/μl
| 1&nbsp;μl
| TCRAC rev 20 pmol/μl
| 1.125 μl
|-
| ACTB probe 10 pmol/μl
| 0.5 μl
| TCRAC probe 10 pmol/μl
| 0.5 μl
|-
| TREC F 10 pmol/μl
| 1 μl
| 2x TaqMan Universal PCR Master Mix
| 12.5 μl
|-
| TREC R 10 pmol/μl
| 1 μl
| colspan="2" rowspan="3" | &nbsp;
|-
| TREC probe 10 pmol/μl
| 0.5 μl
|-
| 2x TaqMan Universal PCR Master Mix&nbsp;
| 10 μl
|}

Assay Mix&nbsp;

Amplicons produced were of 102 bp for ALB2, 256 bp for VD-J and 195 bp for sj-TREC. Standard curves were generated for the different assay where generated as described above. The resulting duplicate Ct values were averaged and used for statistical analysis. The copy number of ALB2, VD-J was calculated