Tox silencer select validated sirna

EXAMPLE 3

5 ng of TOX Silencer Select Validated siRNA (Ambion, Carlsbad, Calif.) was added along with 1.5 μL of TransIT-siQUEST Transfection Reagent (Mirus, Madison, Wis.) per manufacturer's instruction to cultured PBMCs from patients 24 hours after culture was initiated. 72 hours later, RNA was isolated from treated cells using RNAprotect and RNeasy (Qiagen, Valencia, Calif.) per manufacturer's instructions. For cDNA synthesis, total RNA (100 ng) was used for Reverse Transcription (RT) with Superscript II reverse transcriptase (Life Technologies, Gaithersburg, Md.) using oligo dT primers according to the recommendations of the manufacturer. 2 μl of the resulting cDNA was used for each PCR reaction. Quantitative reverse transcription PCR was performed using TaqMan PCR master mix (Applied Biosystems, Foster City, Calif.) together with TaqMan probes and primers (Applied Biosystems) using standard conditions.

As shown in FIG. 6, siRNA knockdown of TOX resulted in decreased TOX expression and increased RUNX3 expression. siRNA knockdown of PLS3 also was found to decrease PLS3 expression.

EXAMPLE 3

5 ng of TOX Silencer Select Validated siRNA (Ambion, Carlsbad, Calif.) was added along with 1.5 μL of TranslT-siQUEST Transfection Reagent (Minis, Madison, Wis.) per manufacturer's instruction to cultured PBMCs from patients 24 hours after culture was initiated. 72 hours later, RNA was isolated from treated cells using RNAprotect and RNeasy (Qiagen, Valencia, Calif.) per manufacturer's instructions. For cDNA synthesis, total RNA (100 ng) was used for Reverse Transcription (RT) with Superscript II reverse transcriptase (Life Technologies, Gaithersburg, Md.) using oligo dT primers according to the recommendations of the manufacturer. 2 μl of the resulting cDNA was used for each PCR reaction. Quantitative reverse transcription PCR was performed using TaqMan PCR master mix (Applied Biosystems, Foster City, Calif.) together with TaqMan probes and primers (Applied Biosystems) using standard conditions.

As shown in FIG. 6, siRNA knockdown of TOX resulted in decreased TOX expression and increased RUNX3 expression. siRNA knockdown of PLS3 also was found to decrease PLS3 expression.

Example 3

5 ng of TOX Silencer Select Validated siRNA (Ambion, Carlsbad, Calif.) was added along with 1.5 μL of TransIT-siQUEST Transfection Reagent (Minis, Madison, Wis.) per manufacturer's instruction to cultured PBMCs from patients 24 hours after culture was initiated. 72 hours later, RNA was isolated from treated cells using RNAprotect and RNeasy (Qiagen, Valencia, Calif.) per manufacturer's instructions. For cDNA synthesis, total RNA (100 ng) was used for Reverse Transcription (RT) with Superscript II reverse transcriptase (Life Technologies, Gaithersburg, Md.) using oligo dT primers according to the recommendations of the manufacturer. 2 μl of the resulting cDNA was used for each PCR reaction. Quantitative reverse transcription PCR was performed using TaqMan PCR master mix (Applied Biosystems, Foster City, Calif.) together with TaqMan probes and primers (Applied Biosystems) using standard conditions.

As shown in FIG. 6, siRNA knockdown of TOX resulted in decreased TOX expression and increased RUNX3 expression. siRNA knockdown of PLS3 also was found to decrease PLS3 expression.