ELISA and real-time PCR

RNA
TR!zol®Reagent (Life technologies) 

Tissue: another small intestine piece (per each srunple) was use for this process. 

1. Add 500µl TRizol® Reagent per each sample (1 mL TRizol® Reagent per 50-100 mg of tissue simple).
2. Homogenize sample with beads (tissuelyzer).
3. Centrifuge your sample at 12,000 x g for 10 minutes at 4°C.

Phase separation 

1. Incubate the homogenized sample for 5 n1inutcs at room temperature to permit complete dissociation of the nucleoprotein  complex.

2. Add l 00µl of chlorofonn per si1nple (0,2 mL of chlorofonn per 1 mL of TRizol® Reagent used for homogenization). Cap the tube securely. 
3. Shake tube vigorously by hand for 15 seconds. 

4. Incubate for 2-3 minutes at room temperature.
5. Centrifuge the sample at 12,000 x g for 15 minutes at 4°C.

Note: The mixture separates into a lower red phenol- chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is `~50% of the total volume. 

6. Remove the aqueous phase of the sample by angling the tube at 45° and pipetting the solution out. A void drawing any of the interphase or organic layer into the pipette when removing the aqueous phase.
7. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure.

RNA Isolation Procedure 
1. Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA. 

RNA precipitation 
2. Add 0.250 mL of 100% isopropanol to the aqueous phase (0.5 mL of 100% isopropanol to the aqueous phase. perl mL of TRizol® Reagent used for homogenization). 
3. Incubate at room temperature for 10 minutes.
4. Centrifuge at 12,000 x g for 10 minutes at 4°C.
Note: The .RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. 
5.Proceed to RNA wash.

RNA wash 
1. Ren1ove the supernatant from the tube, leaving only the RNA pellet.
2. Wash the pellet, witth 0.5 mL of 75% ethanol per sample (1 mL of 75% ethanol per 1 mL of TRJzol® Reagent used in the initial homogenization).
Note: The RNA can be stored in 75% ethanol at least 1 year at -20°C, or at least 1 week at 4°C. 

3. Vortex the sample briefly, then centrifuge the tube at 7500 x g for 5 minutes at 4°C. Discard the wash.
4. Vacuum or air dry the RNA pellet for 5-10 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A_260/280 ratio <1.6.
5.Proceed to RNA resuspension.

RNA resuspension 

1.. Resuspend the RNA pellet in RNase-free water (30µL) by passing the solution up and down several times through a pipette tip. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions.
2. Incubate in a water bath or heat block set at 55-60°C for 10-15 minutes.
3. Proceed to downstream application, or store at -70°C. Samples were stored a -80°C (BOX 4).
 

Real Time PCR Protocol

Following RNA isolation with TRIzol, resuspend RNA pellet in 20 uL RNAse/DNAse free H₂O. 

DNAse Treatment:

Note: These reagents are stored in -80, box A8.

1. Following RNA isolation with TRIzol, resuspend RNA pellet in 20 uL RNAse/DNAse free H20. 
2. Make a master mix of 2 uL 10X DNAse Buffer + 1 uL rDNAse I per sample. Mix 3 uL master mix into sample gently at room temperature. 
3. Incubate on heat block at 37° C for 20-30 minutes
4. Thaw DNAse Inactivation Reagent and vortex. Add 2 uL/sample and mix well well. 
5. Incubate at room temperature for 2 minutes. 
   IMPORTANT: mix gently by hand every ~30 seconds. 
6. Centrifuge at 10,000g for 2 minutes at 4 degrees to remove DNAse Inactivation Reagent. Transfer ~20 uL of the supernatant (RNA) to fresh 1.5 mL tube. 
7. Proceed to Nanodrop to quantify RNA or store RNA at -80°.

cDNA Synthesis:

Note: SuperScript RT kit reagents stored at -20 in RT-PCR box. 

1. If frozen, thaw samples on ice. 
2. Run samples on Nanodrop.
3. Calculate the volume of RNA needed for 500-1000 ng RNA per reaction. Make up the remaining volume with 12 uL H20.
4. cDNA synthesis using SuperScript Reverse Transcriptase II kit. Include a H20 control (use 12 uL of H20 instead of RNA) and a No RT control (use a mix of RNA. Do not add RT)
5. cDNA Reaction (20 uL/sample) in PCR tubes:
   1. 1 uL dNTPs
   2. 2 uL DTT
   3. 0.5 uL Oligos
   4. 0.5 uL RT
   5. 4 uL First-Strand Buffer
   6. 12 uL 500-1000 ng RNA in H20
6. Combine everything together and run on preprogrammed thermalcycler reaction:
   1. 42° C, 50:00
   2. 70° C, 15:00
   3. 4°, infinity
7. Store cDNA at -20° C. 
    

Real Time PCR:

Note: 5^th floor ViiA7 RT-PCR machine only takes 0.2 mL 96 well plates. Do not use the “Fast” reaction option. 

1. Dilute cDNA 1:2 or 1:3 using RNAse/DNAse free H2O (depends on genes of interest).
2. Create master mix for each gene:
   1. For 96-well plate, 20 uL reaction per well:
      1. 10 uL SYBR
      2. 1 uL primer (Note: Qiagen primers contain F+R)
      3. 5 uL H₂O
      4. 4 uL diluted cDNA
   2. For 384-well plate, 9.5 uL reaction per well:
      1. 5 uL SYBR
      2. 0.5 uL primer
      3. 2 uL H₂O
      4. 2 uL diluted cDNA
3. On ice, load SYBR + master mix and cDNA in technical duplicates. Include H20 and No RT controls. 
4. Apply adhesive film and gently spin down or flick plate.