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HSP90B1 siRNA Kit
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Directions for Use
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
Outline of an RNAi Experiment
1. Establish optimal transfection method for your cell type ａnd culture media （use fluorescent-labeled transfection control
duplex）; favored approach is fluorescence microscopy. More than 80% of cells should show dye uptake when examined
4-24 hours after transfection.
2. Demonstrate that RNAi is working using positive control （beta-actin Positive Control duplex）; favored approach is
quantitative RT-PCR. Beta actin should show >70% knockdown 24-48 hours after transfection at 50 nM dose.
3. Test target specific duplexes ａnd perform dose response curve. Selleck Chemicals recommends testing duplexes at 50 nM
concentrations. Knockdown of mRNA levels should be assayed at 24-72 hours after transfection. To limit off-target effects,
routine studies should subsequently be performed using the lowest concentration of RNA duplex that achieves the desired
level of suppression of the target mRNA.
4. Perform RNAi studies using duplexes identified as “effective by >70% reduction in RNA levels”. Selleck Chemicals
recommends that the results of two duplexes against the same target be compared to control for potential off-target effects
and other artifacts.
a. mRNA levels can generally be measured 24-72 hours after transfection.
b. Protein levels can generally be measured at 48-96 hours after transfection, however this may vary depending on the
half-life of the protein studied ａnd cell growth rate.
c. Phenotype studies should parallel protein evaluation.
5. Controls: While examination of non-transfection ａnd mock-transfection cultures （lipid or electroporation alone） are useful,
Selleck Chemicals recommends that control cultures transfected using control RNA duplexes be used for target level
normalization. A randomized sequence （Universal Scrambled Negative Control） duplex is provided for this purpose, which
is not present in human, mouse, or rat.
Transfection Optimization
A. Cell density （% confluence at transfection）: The recommended confluence for most adherent cell types at transfection is
40-60%. We recommend that customers determine the optimal cell density for the particular cell type used in their experiments.
B. Transfection reagent to siRNA ratio: We recommend that you optimize the ratio by adjusting the volume of transfection
reagent within the guidelines of recommended usage by the manufacturer.
The siRNA duplexes in the kit are chemically synthesized short-interfering RNAs, 21-25nt.
The siRNA duplexes have been treated by purifying ａnd annealing.You can dilute siRNA to a to a convenient stock
concentration of 20 μM using sterilized ddH2O or RNase-free H2O.Materials should only be handled with gloves under
RNase-free conditions.Once hydrated, duplexes should be stored at -20°C.Avoid repeated freeze/thaw cycles.Dried RNA
oligonucleotides can be stored in a non-frost free freezer for 1 year at -80°C~ -20°C.
2.
1.
3. Briefly centrifuge tubes or plates containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube.
