Quorum Sensing

2. Approximately 30 mL sheep blood is carefully poured onto
   each Histopaque-1077 column and spun at 2000
   g for
   15 min at room temperature. After centrifugation, the second
   layer from the top (buffy layer), which consists of a thin layer of
   lymphocytes, is recovered using a plastic Pasteur pipette by
   circular motion.


3. The recovered cells are pooled and topped up with sterile PBS
   and centrifuged at 250
   gfor 30 min at room temperature.
   After centrifugation, the supernatant is removed and the cell
   pellet washed in fresh sterile PBS and centrifuged at 250
   gfor
   15 min at room temperature.


4. Total cell number is determined by hemocytometer analysis
   after staining with Trypan Blue. The cells are resuspended in
   an appropriate volume of RNA stabilization buffer to make up
   the cell density to 5
   107 cells/mL and stored at 80 C.


5. Total RNA is extracted from 2
   108 lymphocytes using a
   commercial kit for midi-RNA extraction, following manufac-
   turer’s instructions. Total RNA is then treated with DNase to
   remove genomic DNA contamination, as indicated by the
   manufacturer.


6. For cDNA synthesis, the DNase reaction mix is heated at 70C
   for 10 min to denature both DNaseI and RNA, and chilled on
   ice. DNase-treated total RNA is used as template for cDNA
   synthesis using Superscript III RNase H-Reverse Transcriptase
   (Invitrogen). To 25μL (50–500 ng) of total RNA, 1μL each of
   (25 pmoles) forward primers specific for the ovine antibody
   heavy and light chain constant region of interest (OvCHFOR,
   OvCкFOR, OvCλFOR) are added, and heated at 70C for
   10 min, cooled immediately on ice, and contents collected by
   brief centrifugation. Twelve sets of reactions are set up.


7. Add to the reaction mix, 8μLof5
   first strand buffer, 4μLof
   0.1 M dithiothreitol (DTT), and 1μL of 10 mM dNTP mix
   (2.5 mM each of dATP, dGTP, dTTP, dCTP). Incubate at
   42 C for 2 min before the addition of 1 μL 200 U/μL
   Superscript reverse transcriptase and then incubate at 42C
   for 50 min.


8. Mix the reaction gently with a pipette and incubate for addi-
   tional 50 min at 42C. Stop the reaction by heating the tube at
   70 C for 15 min. Store the cDNA at 20 C until required.


9. To amplify the ovine antibody variable heavy chain, set up
   gradient PCR reactions, by using the following primer pairs:
   (a) OvVH1BACK and mixture of OvJH1LINKFOR, OvJH2-
   LINKFOR, OvJH3LINKFOR, OvJH4LINKFOR.
   (b) OvVH2BACK and mixture of OvJH1LINKFOR, OvJH2-
   LINKFOR, OvJH3LINKFOR, OvJH4LINKFOR.

338 Soumya Palliyil