Material and method

Selection of subjects
The study includes the recruitment of patients from the endemic area of leishmaniasis
performed at Infectious disease research laboratory, Institute of Medical Sciences,
Banaras Hindu University and its field site Kala-azar Medical Research Centre at
Muzaffarpur, Bihar. The study was approved by the Ethical committee of Banaras Hindu
University and written informed consent was obtained from the patients and their
guardians and will be provided with information about the study to be undertaken.
There were two groups- healthy subjects and diseased cases (with two sub-categoriesrelapse cases and fresh cases). The patients were recruited for the treatment on the basis
of the positivity of the results with K-39 strip test. The study had exclusion criteria that
allow the exclusion of patients below the age of 14 years, pregnant women and HIVinfected cases.
Inclusion and Exclusion Criteria
What is being studied and parameters selected?
Details of various procdures and their basis?
The heparinised blood samples were collected from the cases and the controls and the
isolation of the peripheral blood mononuclear cells (PBMC) was done using ficoll by
density gradient centrifugation. The samples were kept in lysis buffer and stored at -80 0C
until further processed. These samples were used for the isolation of RNA by RNeasy
tissue kit (Qiagen GmbH, Hilden, Germany)

and sample quality and integrity was

assessed by nanodrop and agarose (Sigma Aldrich Chemicals, St Louis, MO, USA) gel
electrophoresis. 1000ng of RNA was reverse transcribed using the High Capacity cDNA
synthesis kit (Applied Biosystems, Foster City, CA, USA) as manufacturers instructions.

7500 REAL TIME PCR platform (ABI, Foster City CA, USA) was used together with use
of SYBR green based assay for CD45, IL-10, IFN-gamma, SOCS-1 and SOCS -3.
The relative delta Ct method was used for the quantitation of gene expression and the
expression of genes were normalised against the house keeping gene GAP-DH.
The plate preparation includes the cDNA (5ng/l), 1l of primer/probe 4l of MQ
and 10l of SYBR green master mix (Applied Biosystems, Foster City, CA, USA) with
appropriate no RT and no template controls included in each plate. All samples were run in
duplicate.

Primer sequence used for the experiment

Gene name
IL-10

Primer sequence
F- GTGATGCCCCAAGCTGAGA
R- CACGGCCTTGCTCTTGTTTT

IFN-g

F-TCAGCTCTGCATCGTTTTGG
R- GTTCCATTATCCGCTACATCTGAA

CD45
F-CTGACATCATCACCTAGCAG
R-TGCTGTAGTCAATCCAGTGG
SOCS1

F-TTTTTCGCCCTTAGCGTGA
R-AGCAGCTCGAAGAGGCAGTC

SOCS3

F- AAGGACGGAGACTTCGATTCG
R- AAACTTGCTGTGGGTGACCAT

GAPDH

F- AATGAAGGGGTCATTCATCG
R- AAGGTGAAGGTCGGAGTCAA