OBJECTIVES
– To test whether stimulated B cells differentially express IL-10 upon IVIg treatment. The same samples used in the array will be used in this experiment (samples 11 & 12 will be excluded due to a lack of sample volume).
MATERIALS
– High Capacity RNA-to-cDNA Kit (Ref:4387406, Applied Biosystems)
– TaqMan Universal Master Mix II with UNG (Ref:4440042, Applied Biosystems)
– Human IL-10 Assay Hs00961622-m1 (Applied Biosystems)
– Human 18S Assay Hs99999901-s1 (Applied Biosystems)
PROTOCOLS
A) RT (20160224)
Samples were retrotranscribed according to the following protocol.
| n=1 | MM (n=18+1=19) | |
|---|---|---|
| 2X RT Buffer | 10 mcL | 190 mcL |
| 20X RT Enzyme mix | 1 mcL | 19 mcL |
| Nuclease-free H2O | qsp. 20 mcL | - |
| Sample | up to 9 mcL* | - |
| Total per reaction | 20 mcL | Add 11 mcL per microtube |
200 ng of sample RNA were used in each 20 mcLreaction. The following volumes were added per microtube.
| Identificación | Cuantificación RNA | Volumen. Necesario (200 ng) | |
|---|---|---|---|
| IDENT. MUESTRA | Concentración (ng/mcL) | muestra (mcL) | h2o (mcl) |
| 1 | 137 | 1.4598540145985 | 7.5401459854015 |
| 2 | 150.3 | 1.3306719893546 | 7.6693280106454 |
| 3 | 160.1 | 1.2492192379763 | 7.7507807620237 |
| 4 | 157.2 | 1.2722646310433 | 7.7277353689567 |
| 5 | 49.7 | 4.0241448692153 | 4.9758551307847 |
| 6 | 52.8 | 3.7878787878788 | 5.2121212121212 |
| 7 | 61 | 3.2786885245902 | 5.7213114754098 |
| 8 | 70.1 | 2.8530670470756 | 6.1469329529244 |
| 9 | 75.5 | 2.6490066225166 | 6.3509933774834 |
| 10 | 127.3 | 1.5710919088767 | 7.4289080911233 |
| 13 | 44 | 4.5454545454545 | 4.4545454545455 |
| 14 | 43.4 | 4.6082949308756 | 4.3917050691244 |
| 15 | 84.7 | 2.3612750885478 | 6.6387249114522 |
| 16 | 128.5 | 1.556420233463 | 7.443579766537 |
| 17 | 148.2 | 1.3495276653171 | 7.6504723346829 |
| 18 | 187.1 | 1.0689470871192 | 7.9310529128808 |
| 19 | 133.4 | 1.4992503748126 | 7.5007496251874 |
| 20 | 202.4 | 0.98814229249012 | 8.0118577075099 |
The thermal cycler conditions were as follows, as indicated by Applied (total volume 20 mcL):**
| Step 1 | Step 2 | Step 3 | |
|---|---|---|---|
| Temperature (º C) | 37 | 95 | 4 |
| Time | 60 min | 5 min | Infinite Hold |
C) qPCR (20160224)
Samples were analized in triplets and one blank sample was added to the reaction as negative control.
The following protocol was used:
| N=1 | MM per detector (n=54+3) | |
|---|---|---|
| Taqman Universal Master Mix II | 10 mcL | 570 mcL |
| Taqman Gene Expression Assay | 1 mcL | 57 mcL |
| cDNA template+ H2O | up to 9 mcL* | - |
| Total volume | 20 mcL | Add 11 mcL per well |
1,0 mcL of sample (200ng/20 mcL*1 mcL=10 ng) and 8 mcL of water were added per well. Since 6 wells (n=6+1=7) were analized per sample, a mastermix of 7 mcl of sample+ 56 mcl of water.
The thermal cycler conditions were as follows, as indicated by Applied (total volume 20 mcL):
| UNG incubation (Hold) | Polymerase activation (Hold) | PCR (40 cycles) | |
|---|---|---|---|
| Temperature (ºC) | 50 | 95 | 95 + 60 |
| Time (mm:ss) | 2:00 | 10:00 | 00:15 + 1:00 |
RESULTS
