TaqMan™ Cells-to-CT™ HepatoExpress™ Kit, 400 rxns - FAQs

The TaqMan Cells-to-CT HepatoExpress Kit has not been tested with 3D cultures.

The TaqMan Cells-to-CT HepatoExpress Kit has not been tested with tissue cells in 384-well plates.

No, ezDNase contained in the TaqMan Cells-to-CT HepatoExpress Kit is a double-strand specific DNase and will not degrade cDNA produced in the RT reaction. It is also heat-labile and will be inactivated during the heat kill step of the RT reaction.

With the TaqMan Cells-to-CT HepatoExpress Kit, cells can be washed using warm or cold wash buffer according to user preference. In our evaluation, we found that using warm or chilled wash buffer yielded equivalent results.

Yes, the TaqMan Cells-to-CT HepatoExpress Kit allows for direct RT-qPCR amplification from cell lysates without purification of RNA.

The TaqMan Cells-to-CT HepatoExpress Kit has been tested using the 96-well plate format using the standard 50 μL lysis reaction. If larger well sizes are desired, we recommend scaling up the volume of lysis reaction accordingly and performing a validation experiment.

Yes, the lysate preparation step in the TaqMan Cells-to-CT HepatoExpress Kit protocol is completed at room temperature and does not require any centrifugation steps, making it very automation friendly. We recommend including an orbital shaker on your liquid handler deck for optimal performance.

Yes, the TaqMan Cells-to-CT HepatoExpress Kit includes Express ezDNase which is added to the lysis mixture for removal of gDNA during cell lysis. Express ezDNase is a double-strand specific, heatlabile DNase that will only digest gDNA, making it compatible with the downstream reverse transcription reaction. The enzyme is automatically inactivated during a heat kill step included in the reverse transcription program. To avoid the detection of gDNA, we recommend using a TaqMan Gene Expression Assay specifically designed to span an intron-exon boundary. Such assays are designated with an "_m1" suffix.

gDNA contamination can be identified by performing an RT control reaction without the RT enzyme. The TaqMan Cells-to-CT HepatoExpress Kit contains the SuperScript IV VILO Master Mix No-RT Control which includes all components except for the RT enzyme and can be used for monitoring of gDNA contamination. To avoid the detection of gDNA, we recommend using a TaqMan Gene Expression Assay specifically designed to span an intron-exon boundary. Such assays are designated with an "_m1" suffix.

Yes, lysates from theTaqMan Cells-to-CT HepatoExpress Kit are stable at -80°C for at least 1 year. We have also shown that lysates can go through five freeze/thaw cycles without any loss in signal. It is important to thaw frozen lysates on ice and keep on ice once thawed.

Yes, cDNA generated with the TaqMan Cells-to-CT HepatoExpress Kit is stable at -80°C for at least 1 year. We have also shown that cDNA can go through five freeze/thaw cycles without any loss in signal.

Yes. The lysis reagents within the TaqMan Cells-to- CT HepatoExpress Kit can be purchased in bulk under Cat. No. A65791. The standalone SuperScript IV VILO Master Mix (Cat. No. 11756050), and TaqMan Fast Advanced Master Mix for qPCR (Cat. No. 4444556) can also be purchased separately.

Yes, the TaqMan Fast Advanced Master Mix for qPCR included in the TaqMan Cells-to-CT HepatoExpress Kit supports multiplexed qPCR reactions.

The acceptable range of cell inputs to use with the TaqMan Cells-to-CT HepatoExpress Kit per well of a 96-well plate are:
- Human primary hepatocytes: up to 100,000 cells
- Mouse primary hepatocytes: up to 19,500 cells
- Rat primary hepatocytes: up to 45,000 cells
- HepaRG: up to 100,000 cells
- HepG2: up to 100,000 cells

Yes, the TaqMan Cells-to-CT HepatoExpress Kit allows for the following stopping points:
- After cell lysis: Cell lysates are stable at room temperature for up to 30 minutes, or can be safely stored on ice for up to 2 hours. For long term storage, lysates may be frozen at -80°C for up to 1 year. It is important to thaw frozen lysates on ice and keep on ice once thawed.
- After assembly of the RT reaction: Assembled RT reactions can be stored on ice for up to 9 hours
- After completion of the RT reaction: Completed RT reactions can be stored at -80°C for up to 1 year
- After assembly of the qPCR reaction: Assembled qPCR reactions can be stored at room temperature for up to 72 hours

The two-step RT-qPCR workflow of the TaqMan Cells-to-CT HepatoExpress Kit enables the user to reverse transcribe the entire transcriptome in one RT reaction and then perform separate qPCR reactions for any gene of interest. Two-step RT-qPCR provides the ability to archive cDNA, analyze multiple genes, and gives greater flexibility.

The TaqMan Cells-to-CT HepatoExpress Kit includes the TaqMan Fast Advanced qPCR Master Mix (Cat. No. 4444965). The master mix can be purchased separately if larger qPCR volumes are required by the user.

The TaqMan Cells-to-CT HepatoExpress Kit includes the SuperScript IV VILO Master Mix (Cat. No. 11756050). The master mix can be purchased separately if larger RT volumes are required by the user.

The TaqMan Cells-to-CT HepatoExpress Kit contains TaqMan Fast Advanced qPCR Master Mix and is compatible with the same instruments listed for the TaqMan Fast Advanced qPCR Master Mix, e.g., QuantStudio 3, 5, 6 Pro, 7 Pro, 6 Flex, 7 Flex and 12K Flex instruments. We have tested the TaqMan Cells-to-CT HepatoExpress Kit with the QuantStudio 5 instrument.

Yes, the TaqMan Cells-to-CT HepatoExpress Kit is compatible with TaqMan Array plates and cards. We suggest using the recommended input of cDNA as outlined in the TaqMan Array plates and cards protocols.

Yes, the TaqMan Cells-to-CT HepatoExpress Kit includes the SuperScript IV VILO Master Mix No-RT Control which can be used to determine if the sample contains genomic DNA. For a positive control, we recommend assaying for a highly expressed housekeeping gene.

Yes, the HepatoExpress Lysis Solution in the TaqMan Cells-to-CT HepatoExpress Kit can be stored at room temperature or 4°C.

Yes, every component of the TaqMan Cells-to-CT HepatoExpress Kit is REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)-compliant and non-hazardous. Additionally, there is a significant reduction in plastic waste compared to using column-purified RNA.

The TaqMan Cells-to-CT HepatoExpress Kit consistently produces results that are comparable to those obtained using column-purified RNA over a diverse set of target genes and cell inputs. Comparison data can be found on the product page.

The TaqMan Cells-to-CT HepatoExpress Kit helps provide higher sensitivity than other Cells-to-CT kits when used with primary hepatocytes.

The LysisGuard contained in the TaqMan Cells-to- CT HepatoExpress Kit works in conjuction with the HepatoExpress Lysis Solution to stabilize the sample.

The HepatoExpress Lysis Solution contained in the TaqMan Cells-to-CT HepatoExpress Kit has been formulated to work specifically with primary hepatocytes and with extracellular matrix (ECM) overlays. This solution does not require the use of a stop solution, and is REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)-compliant and free of hazardous materials.

With the TaqMan Cells-to-CT HepatoExpress Kit, up to 40% of the qPCR reaction volume can be cDNA.

With the TaqMan Cells-to-CT HepatoExpress Kit, up to 10% of the RT reaction volume can be cell lysate.

We have tested Thermo Fisher Scientific’s Gibco Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413201) and Corning Matrigel Matrix (Cat. No. 356237) with the TaqMan Cells-to-CT HepatoExpress Kit. We anticipate that the kit will be broadly compatible with other similar ECM products but they should be evaluated by the user.

We have not evaluated the use of smaller lysis reaction volumes with the TaqMan Cells-to-CT HepatoExpress Kit and recommend using the standard 50 μL lysis reaction volume.

We have not evaluated the use of other cells with the TaqMan Cells-to-CT HepatoExpress Kit. We recommend performing a test experiment for any cell types beyond primary hepatocytes, HepaRG, and HepG2.

The TaqMan Fast Advanced qPCR Master Mix can develop particulates that float in solution. Such particulates do not affect kit performance. To minimize the formation of particulates, the master mix should be initially thawed at room temperature and then stored at 4°C for long-term storage.