Thermo Fisher Scientific offers a comprehensive portfolio of lyophilization-compatible (lyo-ready) reverse transcriptases (RTs) and thermostable DNA polymerases, providing:
  • Flexible assay designs while retaining the same functional enzyme performance as with conventional formats
  • Tailor-made solutions for your specific applications, including bulk, custom products, and packaging
  • Higher confidence in results  with low residual DNA contamination (human and bacterial ) of enzymes
  • Assurance of high quality standard of ISO 13485-certified facility

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If you would like to discuss sample availability, please contact us at MDxenzymes@thermofisher.com

Lyo-ready, flexible format

The lyophilized (freeze dried) format of nucleic acid-based assays provides many advantages in terms of stability and shipping costs. Thermo Fisher Scientific has considerable experience in developing lyophilization-compatible enzyme formulations (lyo-ready) that meet high performance requirements for your diagnostic assays.

In conventional formulations of PCR and RT-PCR enzymes, glycerol, at a concentration of up to 50%, is widely used to stabilize enzymes. It serves as a cryo-protecting agent and enables stability of reagents for extended amounts of time. However, even the presence of minute concentrations of glycerol (>0.5%) in the enzyme formulation can interfere with the lyophilization process, thus making most of the conventional enzyme preparations incompatible with lyophilization.  Our lyo-ready enzymes are formulated with dramatically reduced glycerol content, and are made compatible with lyophilization, as stand-alone enzymes or in a reaction mix with other assay components.

Lyo-ready Reverse Transcriptases (RTs)

  Superior enzyme for all RNAs including difficult templates Most referenced RT enzyme in scientific literature
  Lyo-ready SuperScript IV RT Lyo-ready SuperScript III RT
Optimal reaction temperature 50-55oC 50oC
Reaction time 10 min 50 min
Sensitivity Superior Good
Ability to work with degraded or inhibitor-containing RNA Superior

Medium

To receive a sample, contact us at MDxenzymes@thermofisher.com.

RNA sample quality is one of the 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 1).

Figure 1.  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 (red). 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 the average Cq values for each RNA input were calculated.   

Sensitivity, or the ability of RTs to generate cDNA from low input RNA, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript RT is very advantageous for assays where the starting sample amount is limited or the target RNA is at a low concentration in the sample (Figure 2).

Figure 2. High sensitivity cDNA synthesis with lyo-ready reverse transcriptases compared to commercially available glycerol containing RTs. Two step RT-qPCR using 1 ng-10 pg of HeLa total RNA as a template were performed according to the recommended product protocols from each reverse transcriptase vendor.  

Inhibitors, such as trace amount of reagents used in RNA purification, can interfere with 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 the oligo(dT)20 annealing step. Lyo-ready SuperScript RTs function better than other commercially available RTs in the presence of a variety of inhibitors (Figure 3).

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two step RT-qPCR with TFRC gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase supplier.  Inhibitors used: SDS, Guanidinium Hydrochloride, Hemin and Tannic acid.
*TC – Test 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.  Additionally, our comprehensive portfolio of lyo-ready RTs also includes RevertAid (M-MuLV) and Maxima RTs. If you’re interested to learn more, contact us at MDxenzymes@thermofisher.com.

RNA sample quality is one of the 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 1).

Figure 1.  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 (red). 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 the average Cq values for each RNA input were calculated.   

Sensitivity, or the ability of RTs to generate cDNA from low input RNA, is an important attribute for RTs. The high sensitivity of lyo-ready SuperScript RT is very advantageous for assays where the starting sample amount is limited or the target RNA is at a low concentration in the sample (Figure 2).

Figure 2. High sensitivity cDNA synthesis with lyo-ready reverse transcriptases compared to commercially available glycerol containing RTs. Two step RT-qPCR using 1 ng-10 pg of HeLa total RNA as a template were performed according to the recommended product protocols from each reverse transcriptase vendor.  

Inhibitors, such as trace amount of reagents used in RNA purification, can interfere with 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 the oligo(dT)20 annealing step. Lyo-ready SuperScript RTs function better than other commercially available RTs in the presence of a variety of inhibitors (Figure 3).

Figure 3. Resistance to inhibitors. Jurkat total RNA spiked with different inhibitors was used in two step RT-qPCR with TFRC gene specific primers. cDNA reactions were performed according to the recommended protocols from each reverse transcriptase supplier.  Inhibitors used: SDS, Guanidinium Hydrochloride, Hemin and Tannic acid.
*TC – Test 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.  Additionally, our comprehensive portfolio of lyo-ready RTs also includes RevertAid (M-MuLV) and Maxima RTs. If you’re interested to learn more, contact us at MDxenzymes@thermofisher.com.

Lyo-ready DNA Polymerases

  Robust enzyme for all DNAs, including high GC content Value enzyme for commonly used DNA templates
  Platinum Taq  DNA polymerase LibertyTaq DNA polymerase
Hot-start PCR Antibody-based Proprietary
Reactivation time 2 min 0 min
Sensitivity Good Medium
Specificity Good Medium

To receive a sample, contact us at MDxenzymes@thermofisher.com.

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. 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. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder 1.

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

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.

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. 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. NTC = no-template control. DNA ladder: Thermo Scientific ZipRuler Express DNA Ladder 1.

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

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.

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.