Thermo Fisher Scientific offers a comprehensive portfolio of lyophilization-compatible (lyo-ready) reverse transcriptases (RTs) and thermostable DNA polymerases, providing:

  • Flexible assay designs that maximize the same functional enzyme performance as conventional formats
  • Higher confidence in results with minimal risk of bacterial, human or plasmid DNA contamination
  • Assurance of ISO 13485 quality and lot-to-lot consistency
  • Custom solutions for your specific applications, including modified concentration, volume or packaging
  • Lyophilization of your complete assay into a PCR plate or tube, if desired

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Lyo-ready reverse transcriptases (RTs)

  Lyo-ready SuperScript IV RT Lyo-ready SuperScript III RT
Optimal reaction temperature 50–55°C 50°C
Reaction time 10 min 50 min
Sensitivity Superior Good
Inhibitor tolerance Superior Medium
Sample available Yes Yes

Sensitivity, or the ability of RTs to generate cDNA from low RNA input, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript IV RT is advantageous for assays where sample amount is limited or the target RNA is at a low concentration in the sample. The amplification plot (Figure 1) illustrates high sensitivity and linearity of three different formulations of SuperScript IV RT (with glycerol; lyo-ready; lyophilized and reconstituted) across a wide dynamic range of input GAPDH RNA from 1.25 ng to 0.125 fg.

High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RT’s

Figure 1. High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RTs. Two step RT-qPCR for glycerol-containing, lyo-ready, and lyophilized and reconstituted SuperScript IV RT, using 1.25 ng–0.125 fgof GAPDH RNA as template, were performed according to the recommended product protocols.

RNA sample quality is one of major factors determining accuracy and relevance of any downstream analysis. Difficult samples, such as paraffin-embedded samples, buccal swabs, and organ biopsies commonly used for diagnostics and biomedical analysis, present a challenge to obtain high-quality RNA. Integrity of RNA can be affected during handling of samples or through the nucleic acid extraction process.

An efficient RT will reverse transcribe a wide range of RNA targets including those that are degraded during the purification process. Data presented earlier have demonstrated SuperScript IV RT to be robust and efficient with degraded RNA as compared to other commercially available RTs. Here, we show lyo-ready SuperScript IV RT delivers equally efficient cDNA synthesis with degraded RNA as compared to the conventional formulation of SuperScript IV RT (Figure 2).

Highly efficient cDNA synthesis with challenging RNA samples

Figure 2. Highly efficient cDNA synthesis with challenging RNA samples. Two step RT-qPCR reactions of degraded RNA (RIN: 2-3) from human cells were performed with lyo-ready SuperScript IV RT (blue) and conventional SuperScript IV RT with glycerol (orange). Three RT reactions were performed for each input RNA, 10% of the cDNA product was added to TaqMan assays for GAPDH, PPIA, HPRT1 and TBP targets. Three qPCR reactions were performed and average Cq values for each RNA input were calculated.

Inhibitors, such as trace amounts of reagents used in RNA purification, can cause problems during reverse transcription. Inhibitors may also be inherent in the biological sample source such as hemin, found in blood. To test how chemical compounds affect reverse transcription efficiency, various inhibitors were added to Jurkat total RNA prior to oligo(dT)20 annealing step. Figure 3 demonstrates, how lyo-ready SuperScript IV RT performs better than other commercially available RTs in the presence of a variety of inhibitors and retains this ability even after lyophilization and reconstitution.

Resistance to inhibitors

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two -step RT-qPCR with PGK1 gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase vendor. Inhibitors used: SDS, Guanidinium Hydrochloride (GuHCl) and Hemin. NIC – Non Inhibitor Control.

SuperScript IV and SuperScript III lyo-ready RTs offer enhanced sensitivity and reduced reaction times and therefore are recommended for your most demanding RT-qPCR assays.  However, our comprehensive portfolio of lyo-ready reverse transcriptases also includes RevertAid (M-MuLV) and Maxima reverse transcriptases. If you’re interested to learn more, please contact us at MDxenzymes@thermofisher.com.

Sensitivity, or the ability of RTs to generate cDNA from low RNA input, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript IV RT is advantageous for assays where sample amount is limited or the target RNA is at a low concentration in the sample. The amplification plot (Figure 1) illustrates high sensitivity and linearity of three different formulations of SuperScript IV RT (with glycerol; lyo-ready; lyophilized and reconstituted) across a wide dynamic range of input GAPDH RNA from 1.25 ng to 0.125 fg.

High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RT’s

Figure 1. High sensitivity and linearity of cDNA synthesis with lyo-ready, lyophilized and reconstituted SuperScript IV RTs. Two step RT-qPCR for glycerol-containing, lyo-ready, and lyophilized and reconstituted SuperScript IV RT, using 1.25 ng–0.125 fgof GAPDH RNA as template, were performed according to the recommended product protocols.

RNA sample quality is one of major factors determining accuracy and relevance of any downstream analysis. Difficult samples, such as paraffin-embedded samples, buccal swabs, and organ biopsies commonly used for diagnostics and biomedical analysis, present a challenge to obtain high-quality RNA. Integrity of RNA can be affected during handling of samples or through the nucleic acid extraction process.

An efficient RT will reverse transcribe a wide range of RNA targets including those that are degraded during the purification process. Data presented earlier have demonstrated SuperScript IV RT to be robust and efficient with degraded RNA as compared to other commercially available RTs. Here, we show lyo-ready SuperScript IV RT delivers equally efficient cDNA synthesis with degraded RNA as compared to the conventional formulation of SuperScript IV RT (Figure 2).

Highly efficient cDNA synthesis with challenging RNA samples

Figure 2. Highly efficient cDNA synthesis with challenging RNA samples. Two step RT-qPCR reactions of degraded RNA (RIN: 2-3) from human cells were performed with lyo-ready SuperScript IV RT (blue) and conventional SuperScript IV RT with glycerol (orange). Three RT reactions were performed for each input RNA, 10% of the cDNA product was added to TaqMan assays for GAPDH, PPIA, HPRT1 and TBP targets. Three qPCR reactions were performed and average Cq values for each RNA input were calculated.

Inhibitors, such as trace amounts of reagents used in RNA purification, can cause problems during reverse transcription. Inhibitors may also be inherent in the biological sample source such as hemin, found in blood. To test how chemical compounds affect reverse transcription efficiency, various inhibitors were added to Jurkat total RNA prior to oligo(dT)20 annealing step. Figure 3 demonstrates, how lyo-ready SuperScript IV RT performs better than other commercially available RTs in the presence of a variety of inhibitors and retains this ability even after lyophilization and reconstitution.

Resistance to inhibitors

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two -step RT-qPCR with PGK1 gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase vendor. Inhibitors used: SDS, Guanidinium Hydrochloride (GuHCl) and Hemin. NIC – Non Inhibitor Control.

SuperScript IV and SuperScript III lyo-ready RTs offer enhanced sensitivity and reduced reaction times and therefore are recommended for your most demanding RT-qPCR assays.  However, our comprehensive portfolio of lyo-ready reverse transcriptases also includes RevertAid (M-MuLV) and Maxima reverse transcriptases. If you’re interested to learn more, please contact us at MDxenzymes@thermofisher.com.

Lyo-ready Taq DNA polymerases

  Platinum II Taq DNA polymerase Platinum Taq DNA polymerase LibertyTaq DNA polymerase
Hot-start PCR Antibody-based Antibody-based Proprietary
Reactivation time 30 sec to 2 min 2 min 0 min
Sensitivity Superior Good Medium
Specificity Superior Good Medium
Sample availability Yes Yes Yes

Platinum Taq DNA polymerase uses stringent antibody-based hot-start technology, which enables detection of low-abundance DNA targets with high accuracy. Similarly, lyo-ready Platinum Taq CG DNA polymerase offers the same great performance as Platinum Taq DNA polymerase, but in a formulation compatible with lyophilization (Figure 4).

High sensitivity with lyo-ready Platinum Taq, CG DNA Polymerase

Figure 4. High sensitivity with lyo-ready Platinum Taq CG DNA Polymerase. Lyo-ready Platinum Taq CG and Platinum Taq DNA Polymerases were used to amplify a 199 bp fragment of human DNA (A) and a 639 bp fragment of E. coli DNA (B) from varying amounts of template DNA in duplicate assays. The lyo-ready enzyme formulation offered sensitivity, specificity, and yields comparable to those of the standard formulation of Platinum Taq polymerase. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder.

Lyo-ready Platinum Taq and LibertyTaq Hot start DNA polymerases are a strong choice for qPCR based assays. These enzymes offer fast activation, robust, sensitive and specific amplification across a wide dynamic range.

Efficient and reproducible qPCR assays

Figure 5. Efficient and reproducible qPCR assays. Lyo-ready LibertyTaq (blue curves) and Invitrogen Platinum Taq (red curves) DNA Polymerases were evaluated for their performance in qPCR using Applied Biosystems TaqMan Assays for human PPP1CA and varying amounts of human input DNA. Equally efficient and sensitive amplification was achieved with both DNA polymerases. No amplification was observed in no-template controls, indicating that, based on this detection method, formulations are free of contaminating human DNA. qPCR reaction efficiency is in the range of 90-110% with a determination coefficient R2 ≥0.990.

Stringent manufacturing process and a proprietary low glycerol formulation allows lyo-ready Platinum Taq and LibertyTaq DNA polymerases to exhibit high stability even under non-optimal shipping and storage conditions. The stability of DNA polymerase after multiple freeze/thaw cycles ensures product quality and performance in nucleic acid based assays.

Stability of LibertyTaq DNA Polymerase under different shipping conditions

Figure 6. Stability of LibertyTaq DNA Polymerase under different shipping conditions. To mimic different shipping and storage conditions, Lyo-ready LibertyTaq DNA Polymerase was subjected to 20 freeze/thaw cycles prior to its use in E. coli 23S TaqMan Assays (blue curves). Performance was compared to that of lyo-ready LibertyTaq DNA Polymerase stored at –20°C (red curves). Multiple freezing and thawing cycles did not affect the enzyme’s performance.

Platinum Taq DNA polymerase uses stringent antibody-based hot-start technology, which enables detection of low-abundance DNA targets with high accuracy. Similarly, lyo-ready Platinum Taq CG DNA polymerase offers the same great performance as Platinum Taq DNA polymerase, but in a formulation compatible with lyophilization (Figure 4).

High sensitivity with lyo-ready Platinum Taq, CG DNA Polymerase

Figure 4. High sensitivity with lyo-ready Platinum Taq CG DNA Polymerase. Lyo-ready Platinum Taq CG and Platinum Taq DNA Polymerases were used to amplify a 199 bp fragment of human DNA (A) and a 639 bp fragment of E. coli DNA (B) from varying amounts of template DNA in duplicate assays. The lyo-ready enzyme formulation offered sensitivity, specificity, and yields comparable to those of the standard formulation of Platinum Taq polymerase. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder.

Lyo-ready Platinum Taq and LibertyTaq Hot start DNA polymerases are a strong choice for qPCR based assays. These enzymes offer fast activation, robust, sensitive and specific amplification across a wide dynamic range.

Efficient and reproducible qPCR assays

Figure 5. Efficient and reproducible qPCR assays. Lyo-ready LibertyTaq (blue curves) and Invitrogen Platinum Taq (red curves) DNA Polymerases were evaluated for their performance in qPCR using Applied Biosystems TaqMan Assays for human PPP1CA and varying amounts of human input DNA. Equally efficient and sensitive amplification was achieved with both DNA polymerases. No amplification was observed in no-template controls, indicating that, based on this detection method, formulations are free of contaminating human DNA. qPCR reaction efficiency is in the range of 90-110% with a determination coefficient R2 ≥0.990.

Stringent manufacturing process and a proprietary low glycerol formulation allows lyo-ready Platinum Taq and LibertyTaq DNA polymerases to exhibit high stability even under non-optimal shipping and storage conditions. The stability of DNA polymerase after multiple freeze/thaw cycles ensures product quality and performance in nucleic acid based assays.

Stability of LibertyTaq DNA Polymerase under different shipping conditions

Figure 6. Stability of LibertyTaq DNA Polymerase under different shipping conditions. To mimic different shipping and storage conditions, Lyo-ready LibertyTaq DNA Polymerase was subjected to 20 freeze/thaw cycles prior to its use in E. coli 23S TaqMan Assays (blue curves). Performance was compared to that of lyo-ready LibertyTaq DNA Polymerase stored at –20°C (red curves). Multiple freezing and thawing cycles did not affect the enzyme’s performance.

Lyo-ready Proofreading DNA polymerases

Product Platinum SuperFi DNA Polymerase Phusion Hot Start II High-Fidelity DNA Polymerase
Fidelity vs. Taq polymerase >100X 52X
Hot-start PCR Antibody-based Antibody-based
Target length ≤20 kb ≤20 kb
Extension rate 15-30 sec/kb 15-30 sec/kb
TaqMan probe-compatible No No
Inhibitor tolerance ++++ +++
Multiplexing Yes Yes
Sample availability Yes Yes

Invitrogen Platinum SuperFi DNA polymerase is engineered with a DNA-binding domain exhibiting high processivity and increased resistance to common PCR inhibitors such as Guanidinium Hydrochloride and Urea. Lyo–ready Platinum SuperFi DNA polymerase retains reliable performance in presence of the inhibitors.

Reliable performance in presence of common PCR inhibitors

Figure 7. Reliable performance in presence of common PCR inhibitors. Amplification of ~2 kb E.coli gDNA fragment using lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 ng gDNA and 2 – no inhibitor, 3 – 45 mM guanidine hydrochloride, 4 – 200 mM urea. 1 – no template control.

Lyo-ready Platinum SuperFi DNA polymerase combines performance properties attributed to standard glycerol formulation and offers that same performance in lyo-ready and lyophilized and reconstituted state.

Reliable performance across various formats

Figure 8. Reliable performance across various formats (glycerol, lyo-ready and lyophilized and reconstituted). Amplification of 666 bp E.coli gDNA fragment using lyo-ready Platinum SuperFi, lyophilized and reconstituted lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 – 10 ng DNA, 2 – 1 ng DNA, 3 – 0.1 ng DNA.

Invitrogen Platinum SuperFi DNA polymerase is engineered with a DNA-binding domain exhibiting high processivity and increased resistance to common PCR inhibitors such as Guanidinium Hydrochloride and Urea. Lyo–ready Platinum SuperFi DNA polymerase retains reliable performance in presence of the inhibitors.

Reliable performance in presence of common PCR inhibitors

Figure 7. Reliable performance in presence of common PCR inhibitors. Amplification of ~2 kb E.coli gDNA fragment using lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 ng gDNA and 2 – no inhibitor, 3 – 45 mM guanidine hydrochloride, 4 – 200 mM urea. 1 – no template control.

Lyo-ready Platinum SuperFi DNA polymerase combines performance properties attributed to standard glycerol formulation and offers that same performance in lyo-ready and lyophilized and reconstituted state.

Reliable performance across various formats

Figure 8. Reliable performance across various formats (glycerol, lyo-ready and lyophilized and reconstituted). Amplification of 666 bp E.coli gDNA fragment using lyo-ready Platinum SuperFi, lyophilized and reconstituted lyo-ready Platinum SuperFi, standard (with glycerol) Platinum SuperFi DNA polymerases. 50 µL reaction mixes contained 1 – 10 ng DNA, 2 – 1 ng DNA, 3 – 0.1 ng DNA.

Lyo-ready master mixes

Applied Biosystems qPCR master mixes are available in a lyo-ready format on a custom basis to our commercial partners for genotyping, gene expression, and copy number variation applications. Further customization is available, and all master mixes are available with or without a passive reference dye. If you are interested in a lyo-ready master mix, contact us, and we will work with you to deliver the right formulation for your unique assay.

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Our low-glycerol, lyophilization-ready qPCR master mixes are one-tube solutions that are ready to be mixed with primers, probes, and excipients, for the subsequent lyophilization step. There is no need for time-consuming development, optimization, and sourcing of multiple separate qPCR reaction components. These reagents retain the reproducibility, sensitivity, and specificity required for diagnostic assays. Once lyophilized, the assays demonstrate little to no loss of sensitivity or specificity, and have been shown to reproducibly detect single-copy targets per reaction and provide a linear dynamic range over 7 logarithmic units.

Comparison of qPCR amplification curves

Figure 9. Comparison of qPCR amplification curves using Applied Biosystems TaqMan® Lyo-ready 1-Step Master Mix and Applied Biosystems TaqMan® Fast Virus (FV) 1-Step Master Mix. The Applied Biosystems VetMAX Xeno™ Internal Positive Control was used as the target, in a dilution series over 7 orders of magnitude. The TaqMan Lyo-ready 1-Step Master Mix maintains or exceeds the performance of TaqMan FV 1-Step Master Mix in Ct and fluorescence (ΔRn) values. The TaqMan Lyo-ready 1-Step Master Mix can be formulated with Applied Biosystems ROX passive reference dye (as shown here) or without the passive reference dye.

TaqMan lyo-ready master mixes are turnkey formulations that can be inserted into the lyophilization process with little to no optimization. They yield the sensitivity and linear dynamic range you have come to expect from Applied Biosystems qPCR master mixes, and post-lyophilization results are similar to those achieved pre-lyophilization. To demonstrate performance before and after lyophilization, the TaqMan Lyo-ready 1-Step Master Mix was first combined with excipients to form a pre-lyo master mix, and then lyophilized on an FTS Systems LyoStar II system. The performance of the TaqMan Lyo- Ready 1-Step Master Mix is retained with or without the added excipients, and after lyophilization (Figure 8).

Performance comparison of lyoready, pre-lyo, and lyophilized master mixes

Figure 10. Performance comparison of lyoready, pre-lyo, and lyophilized master mixes to that of TaqMan Lyo-ready 1-Step Master Mix, using a TaqMan XenoRNA™ Gene Expression Assay. Ct values, PCR efficiency, and R2 are consistent across the three variations of master mix.

Lyophilized assays minimize stringent requirements for cold-chain storage and shipping, dramatically reducing costs to molecular diagnostics developers and their customers. Lyophilized assays also exhibit higher stability, thus reducing waste due to reagent and assay expiration. Figure 9 shows consistent stability of three batches of lyophilized assay using TaqMan Lyo-ready 1-Step Master Mix for four targets tested.

Stability testing of different batches of TaqMan Lyo-Ready 1-Step Master Mix
 Click to enlarge

Figure 11. Stability testing of different batches of TaqMan Lyo-ready 1-Step Master Mix. Three batches of a lyophilized multiplex arbovirus assay using TaqMan Lyo-ready 1-Step Master Mix were stored at ambient temperature (24°C) and tested for their stability at regular intervals. Stability results for all batches of the lyophilized 1-step master mix with four targets showed that the ΔCt values (Ct test point – Ct time 0) remained within the specified metric of ±1 over 146 weeks (2.8 years).

Our low-glycerol, lyophilization-ready qPCR master mixes are one-tube solutions that are ready to be mixed with primers, probes, and excipients, for the subsequent lyophilization step. There is no need for time-consuming development, optimization, and sourcing of multiple separate qPCR reaction components. These reagents retain the reproducibility, sensitivity, and specificity required for diagnostic assays. Once lyophilized, the assays demonstrate little to no loss of sensitivity or specificity, and have been shown to reproducibly detect single-copy targets per reaction and provide a linear dynamic range over 7 logarithmic units.

Comparison of qPCR amplification curves

Figure 9. Comparison of qPCR amplification curves using Applied Biosystems TaqMan® Lyo-ready 1-Step Master Mix and Applied Biosystems TaqMan® Fast Virus (FV) 1-Step Master Mix. The Applied Biosystems VetMAX Xeno™ Internal Positive Control was used as the target, in a dilution series over 7 orders of magnitude. The TaqMan Lyo-ready 1-Step Master Mix maintains or exceeds the performance of TaqMan FV 1-Step Master Mix in Ct and fluorescence (ΔRn) values. The TaqMan Lyo-ready 1-Step Master Mix can be formulated with Applied Biosystems ROX passive reference dye (as shown here) or without the passive reference dye.

TaqMan lyo-ready master mixes are turnkey formulations that can be inserted into the lyophilization process with little to no optimization. They yield the sensitivity and linear dynamic range you have come to expect from Applied Biosystems qPCR master mixes, and post-lyophilization results are similar to those achieved pre-lyophilization. To demonstrate performance before and after lyophilization, the TaqMan Lyo-ready 1-Step Master Mix was first combined with excipients to form a pre-lyo master mix, and then lyophilized on an FTS Systems LyoStar II system. The performance of the TaqMan Lyo- Ready 1-Step Master Mix is retained with or without the added excipients, and after lyophilization (Figure 8).

Performance comparison of lyoready, pre-lyo, and lyophilized master mixes

Figure 10. Performance comparison of lyoready, pre-lyo, and lyophilized master mixes to that of TaqMan Lyo-ready 1-Step Master Mix, using a TaqMan XenoRNA™ Gene Expression Assay. Ct values, PCR efficiency, and R2 are consistent across the three variations of master mix.

Lyophilized assays minimize stringent requirements for cold-chain storage and shipping, dramatically reducing costs to molecular diagnostics developers and their customers. Lyophilized assays also exhibit higher stability, thus reducing waste due to reagent and assay expiration. Figure 9 shows consistent stability of three batches of lyophilized assay using TaqMan Lyo-ready 1-Step Master Mix for four targets tested.

Stability testing of different batches of TaqMan Lyo-Ready 1-Step Master Mix
 Click to enlarge

Figure 11. Stability testing of different batches of TaqMan Lyo-ready 1-Step Master Mix. Three batches of a lyophilized multiplex arbovirus assay using TaqMan Lyo-ready 1-Step Master Mix were stored at ambient temperature (24°C) and tested for their stability at regular intervals. Stability results for all batches of the lyophilized 1-step master mix with four targets showed that the ΔCt values (Ct test point – Ct time 0) remained within the specified metric of ±1 over 146 weeks (2.8 years).

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