Fig. 1. a. RT-PCR detection of brain specific transcript, SOD1 (500 bp) in human fibroblast cultured cells. Poly (A) containing total RNA has prepared, reverse transcribed to cDNA from 1 μg of RNA, amplified with SOD1 specific primers for 30 cycles. The RT-PCR products are subjected to electrophoresis through 2% agarose gel in 1X TBE buffer pH 8.3. The gel images are documented with the help of Gel Documentation and Image Analysis System. Lane M: low mass DNA ladder from 100 to 2000 bp. Lane 1 and 2: RT-PCR products for 50 year normal female and male. Lane 3: product for 2 year normal child. Lane 4 and 5: products for 1 year DS female and male child. Lane 6: nucleic acid free negative control. Lane 7: β-Actin positive control. b. Hae III enzyme digestion of 500 bp RT-PCR product at 37 °C for 1 hour generating 4 fragments: Lane M: low mass DNA ladder from 100 to 2000 bp. Lane 1 and 5: fragments for 1 year DS male and female respectively. Lane 2: fragments for 2 year normal male. Lane 3 and 4: fragments for 50 year male and female respectively. c. RT-PCR detection of SOD2 gene (325 bp) in human fibroblast cultured cells. Lane M: low mass DNA ladder from 100 to 2000 bp. Lane 1 and 2: RT-PCR products for 50 year normal female and male respectively. Lane 3: product for 2 year normal child. Lane 4 and 5: product for 1 year DS female and male child respectively. Lane 6: nucleic acid free negative control. Lane 7: β-Actin positive control. d. RT-PCR detection of anti-oxidant gene GPx (628 bp) in human fibroblast cultured cells. Lane M: low mass DNA ladder from 100 to 2000 bp. Lane 1 and 2: RT-PCR products for 50 year normal female and male. Lane 3: product for 2 year normal child. Lane 4 and 5: products for 1 year DS female and male child. Lane 6: nucleic acid free negative control. Lane 7: β-Actin positive control. e. RT-PCR detection of anti-oxidant gene CAT (493 bp) in human fibroblast cultured cells. Lane M: low mass DNA ladder from 100 to 2000 bp. Lane 1 and 2: RT-PCR products for 50 year normal female and male. Lane 3: product for 2 year normal child. Lane 4 and 5: products for 1 year DS female and male child. Lane 6: nucleic acid free negative control. Lane 7: β-Actin positive control.

Fig. 2. a. Differential gene expression of SOD1 with internal positive control β-Actin in normal (control), aged and DS affected human fibroblast cultured cells. b. Differential gene expression of SOD2 with internal positive control β-Actin in normal (control), aged and DS affected human fibroblast cultured cells. c. Differential gene expression of GPx with internal positive control β-Actin in normal (control), aged and DS affected human fibroblast cultured cells. d. Differential gene expression of CAT with internal positive control β-Actin in normal (control), aged and DS affected human fibroblast cultured cells.

Fig. 3. Western blot of human fibroblast cell lysate protein (16μg total protein) SOD1 (16 kD), GPx (23 and 92 kD), SOD2 (40 kD), Actin (43 kD) and CAT (64 kD) of normal, aged and DS male and female respectively. Lane 1 shows western protein standards from 20 to 220 kD. Lane 2 and 3 show protein bands for 50 year aged normal female and male respectively. Lane 4 shows protein bands of 2 year male (control). Lane 5 and 6 show protein bands of 1 year aged DS female and male respectively.

RT-PCR amplification of anti-oxidant mRNA genes in normal, aging and DS affected human fibroblast cultured cells

Quantitation of anti-oxidant gene expression in normal, aging and trisomic fibroblast cultured cells

Average of three determinations (Mean ± SD).

Normal, aging and DS based differential anti-oxidant gene expression in relation to endogenous positive gene β-actin

Ratios of copper zinc superoxide dismutase and manganese superoxide dismutase with respect to glutathione peroxidase and catalase