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Protocol 15.1: Monitoring solid organ transplant recipients for rejection

A. URINE, BLOOD, AND BIOPSY PROCESSING_

1. Urine 1.1 Urine collection

1.1.1 Urines are to be placed on ice and/or into the refrigerator immediately after obtaining sample.

1.1.2 Time collected needs to be noted on the specimen.

1.1.3 Samples need to be processed up at our earliest opportunity.

1.1.4 Log volume and appearance (cloudy, clear).

1.2 Urine processing (process within 1 h of collection).

1.2.1 Spin the total amount of urine for 5 min at 2,200 rpm.

1.2.2 Remove 1 ml urine supernatant and transfer to a 2 ml freezing vial labeled with the accession number only and identify it as urine supernatant. Freeze at -80 in box provided for viral detection study.

1.2.3 Resuspend the pellet in 1 x PBS and transfer to a 1.5 ml capped microcentrifuge tube. Spin for 2.5 min at 3,400 rpm.

1.2.4 Following the wash, remove the supernatant and resuspend in 0.5 ml 1 x PBS.

1.2.5 Mix 20 ^L of this suspension, 20 ^L Trypan Blue and 60 ^L 1 x PBS. Count the number of leukocytes present. Note the presence of epithelial cells, bacteria, etc.

1.2.6 Pellet the remaining suspension by spinning at 1,000 rpm, aspirate supernatant, either add 0.5 ml RNA later and freeze at 20°C or proceed with RNA extraction as discussed later.

2. Whole blood 2.1

Whole blood collection Three 7 ml ACD tubes are to be drawn, mixed immediately, and kept at room temperature until picked up. Check tubes for a complete name, date drawn, and second identifier, such as DOB. If there is any danger of the specimen reaching temperatures less than 60°F, pack the tubes inside a double container, preferably Styrofoam.

Specimen logging

Note the temperature of the specimen if other than RT when received. Record total volume prior to spinning; note the packed cell volume (PCV) and any hemolysis after spinning.

Specimen processing Pour all blood into a 50 ml conical tube. Note the volume then fill to the 50 mark with RPMI with 2% FCS. Mix briefly and spin at 2,200 rpm for 5 min with the brake in the OFF position.

Aspirate the supernatant to within 1/4 inch of the buffy coat. Note PCV prior to doubling the volume in increments of 10 with RPMI with 2% FCS. Mix well by rocking to break up any leukocyte clumps.

Overlay 5 ml Ficoll with 10 ml red cell suspension in a 15 ml conical tube. Spin for 30 min at 1,400 rpm with the brake on LOW.

Remove the mononuclear interface to a 15 ml conical tube containing RPMI as soon as the centrifuge stops. Top the tube off with RPMI and spin at 1,600 rpm for 5 min. Repeat this wash. Resuspend the pellet in 2 ml cold 1 x PBS and count. Specimens containing a large number of platelets should be rewashed. Note viability on the worksheet. Pellet the cells by spinning for 2.5 min at 3,400 rpm aspirate PBS and either resuspend in 0.5 ml RNA Later and freeze at -20°C or proceed with the RNA extraction.

3. Biopsy Tissue is either stored in RNA Later at -20°C

until it is processed or processed immediately

B. RNA ISOLATION, AND DNASE TREATMENT PROTOCOL

Using RNAqueous™ kit and the manual provided by Ambion

Buffer preparation: 64% ethanol: Add 38.4 ml 100% ACS grade ethanol to the bottle that contains 21.6 ml nuclease-free water (provided in the kit) for a final concentration of 64% ethanol. Wash solution 2/3: dilute the solution with 64% ml 100% ACS grade ethanol before use

1. Rinse cells once in PBS by gentle resuspension and centrifugation, to remove any RNA Later (Qiagen) solution

2. Remove PBS and add 300 uL lysis/binding solution per 106 to 107 cells. If the lysate is extremely viscous, further dilute with lysis/binding solution

3. Add an equal volume of 64% ethanol and mix well by repeated pipetting

4. Insert an RNAqueous™ filter cartridge (Ambion) into one of the RNase-free collection tubes supplied. Apply the lysate/ethanol mixture to the filter. 700 \iL is the maximum volume that can be applied at one time. It is not recommended to exceed 1,800 \iL lysate/ethanol mixture per filter

5. Centrifuge for 1 min, discard the flow-through, and reuse the tube for the washing steps.

6. Wash the filter cartridge with 700-pL wash solution #1 (provided in kit), centrifuge for 1 min, discard the flow-through, and reuse the tube for the subsequent washes

7. Wash the filter cartridge twice with 500-pL wash solution #2/3, centrifuge for 1 min, and discard the flow-through, centrifuge for an extra 2 min to remove any traces of wash solution

8. Transfer filter cartridge to fresh collection tube. Add 25 pL of elution solution to the center of the filter. Incubate the tube with cartridge in a heat block set at 65-70°C, for 10 min. Recover the elute by centrifuging for 1 min

9. To maximize recovery of RNA, repeat step 7, except do not transfer the cartridge to a fresh collection tube

10. To measure optical density, take 10 pL of the eluted RNA and add 90 uL of DEPC water. Measure the absorbance at 260 nm using the GeneQuant Pro spectrophotometer (Amersham Pharmacia Biotechnology)

DNase treatment using DNA-free™ kit and the manual provided by

Ambion.

Use individually wrapped tubes and tips.

1. Add 0.1 volume** of 10X DNase 1 buffer and 1 uL of DNase 1-(2 units) to the RNA. Mix gently and incubate at 37°C for 20-30 min

2. Add 0.1 volume or 5 pL, whichever is greater, of the resuspended DNase inactivation reagent to the sample and mix well

3. Incubate for 2 min at room temperature.

4. Centrifuge the tube for 1 min to pellet the DNase inactivation reagent

5. RNA should be stored at -80°C until used

** Volume means 0.1X the amount of RNA

C. REAL-TIME PCR PROTOCOL_

1. TaqMan® probes and primers.

a. Probes and primers for the gene of interest are designed using the Primer Express® software (PE Applied Biosystems, CA, USA).

b. All primer pairs were designed to produce amplicons smaller than 150 bp

2. Preparing TaqMan® probes from ABI already resuspended at 100 pM.

a. A dilution of 1/20 is made to give a 5pM solution b. Aliquot the probe solution and store in the dark at 20°C. It is not recommended to thaw and freeze more than twice

3. The primers arrive lyophilized with the amount given on the tube in pmols (such as 150.000 pmol which is equal to 150 nmol).

a. If X nmol of primer is resuspended in XpL of H2O, the resulting solution is

1 mM

b. Freeze this stock solution in aliquots c. When the 1 mM stock solution is diluted 1/100, the resulting working solution will be 10 pM

d. To get the recommended 50-900 nM final primer concentration in 50 pL

reaction volume, 0.25-4.50 |L should be used per reaction (2.5 |L for 500 nM final concentration)

The pre-developed (PDAR) primers and probes are supplied as a mix in one tube. It is recommended to use 2.5 |L in a 50 |L reaction volume.

4. Setting up one-step TaqMan® reaction

Using the TaqMan® EZ RT-PCR kit (PE Applied Biosystems) and the manual provided a) Prepare a reagent mix containing all the PCR components except target RNA. Preparing a reagent mix is recommended in order to increase the accuracy of the results

Reagent mix

RNase-free H2O

5X TaqMan® EZ buffer

Manganese acetate (25 mM)

rTth DNA Polymerase (2.5 U/|L)

Total mix

Target RNA

Volume for one sample Final concentration

14.5 uL [18 uL For 10.0 ||L 6.0 |L 1.5 |L 1.5 |L 1.5 |L 1.5 |L 2.5 |L 2.5 |L 1.0 |L 0.25 |L 0.5 |L 45 |L 5 |L

PDAR 18S MIX] 1X

3 mM 300 |M 300 |M 300 |M 600 |M 500 nM 500 nM 100n M 0.1 U/|L 0.01 U/|L

* If a PDAR is used, 2.5 |L of primer + probe mix used b) Amplification of the target genes tested and the 18S RNA is to be performed in duplicate in the same plate c) In each plate a non-template control (for each reagent mixture) will be run to test for any primer-dimmer or contamination d) In the case of using an absolute standard curve or relative standard curve, the standard must be run in duplicate in each plate e) The program consists of heating at 50°C for 2 min, 60°C for 30 min, and 95°C for 5 min, followed by 40 cycles of a two-

stage temperature profile of 94°C for 20 sec and 62°C for 1 min f) Accumulation of the PCR products is detected by directly monitoring the increase in fluorescence of the reporter dye g) Data points collected in this manner are analyzed at the end of thermal cycling

5. Analyzing and interpreting the results a) Save the run before it starts by giving it a name (not as untitled). Also at the end of the run, first save the data before starting to analyze b) The choice of dye component should be made correctly before data analysis. Example; if the probe is labeled with FAM and VIC is chosen there will be some result but the wrong one c) When analyzing the data ensure that the default setting for baseline is 3-15. If any Ct value is <15, the baseline should be changed accordingly (the baseline stop value should be 1-2 smaller than the smallest Ct value)

d) A threshold for the amplification of each gene of interest is set by drawing a line that intersects the exponential phase of the logarithmic amplification curves for all samples being analyzed for expression of target gene (A threshold line should be drawn above the background noise and below the plateau region). The cycle number at which the threshold line intersects the linear curve for each sample is used to determine the threshold cycle (Ct) value e) Rn+ is the Rn value of a reaction containing all components; Rn- is the Rn value of an unreacted sample (baseline value or the value detected in NTC). ARn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR

Final quantification can be done by any method. For the purposes of renal transplant monitoring relative quantification methods are satisfactory (see Chapters 2, 3, 6, and 7).

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