superscript iv reverse transcriptase— top iv … · with random hexamers according to the...

SuperScript IV Reverse Transcriptase— top IV reasons to love the best Super RT yet

Reason #2: SuperScript IV RT delivers up to 100x higher yield with degraded samplesThe ideal RT will reverse-transcribe even the most difficult types of RNA, such as RNA samples degraded during the purification process (which results in even fewer copies of full-length RNA transcripts). SuperScript IV RT is robust and sensitive enough to detect degraded RNA (RIN 1–3), making it a superior choice for cDNA synthesis.

Figure 2. 100-fold higher yield with degraded RNA using SuperScript IV RT. (A) A 1.2% Invitrogen™ E-Gel™ agarose gel with 500ng total RNA from different samples. High-quality RNA (RIN >8) with intact ribosomal RNA is shown. Other RNA samples had a RIN score between 1 and 3 as determined on an Agilent Bioanalyzer™ instrument. (B) RT-qPCR of degraded total RNA. Degraded RNA (100ng) was used in a 20μL SuperScript IV RT reaction with random hexamers according to the provided protocol. Reverse transcriptases from other vendors were used according to the manufacturers’ recommended protocols. From each RT reaction, 10% of the cDNA was added to Invitrogen™ EXPRESS qPCR Supermix, Universal (Cat. No. 11785-01K). Applied Biosystems™ TaqMan™ Assays for the gene targets are indicated in the figures. Ct values were normalized to SuperScript IV RT using the equation: Normalized RT sensitivity = 2^(Ct SSIV – Ct competitor). SSIV: SuperScript IV RT. SSIII: SuperScript™ III RT.

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Reason #1: SuperScript IV RT reaction takes as little as 10 minutesThe more processive an enzyme, the longer the cDNA that can be synthesized and the faster it can make full-length cDNA. Only SuperScript IV RT can synthesize up to 9kb cDNA in just 10 minutes, while the rest of the commercially available RTs failed to synthesize cDNA up to 3kb in 30–60 minutes using their recommended protocols (Figure 1).

Figure 1. Fast cDNA synthesis. 500ng of Invitrogen™ Ambion™ Millennium™ RNA Markers (Cat. No. AM7150) was used in a 10μL SuperScript IV RT reaction with oligo(dT)20 according to the provided protocol. Reverse transcriptases from other vendors were tested following the manufacturers’ recommended protocols, except a 10 min reaction time was used. First-strand cDNA was resolved by alkaline gel electrophoresis and stained using Invitrogen™ SYBR™ Gold Nucleic Acid Gel Stain (Cat. No. S11494). NaOH was used to hydrolyze all RNA, resulting in visualization of only cDNA.

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Invitrogen™ SuperScript™ IV Reverse Transcriptase (RT) is the newest member of the Invitrogen™ SuperScript™ RT product line, known for its quality and reliability in cDNA synthesis. Here are the top IV reasons why this RT performs well.

Reason #4: SuperScript IV RT works every timeInhibitory components frequently found in RNA samples can interfere with cDNA synthesis and give false-negative RT-PCR and RT-qPCR results. SuperScript IV RT shows significant resistance to contaminating inhibitors as compared to Invitrogen™ SuperScript™ III RT and commercially available RTs (Figure 4). This feature helps to obtain robust results, even with lower-purity samples like formalin-fixed, paraffin-embedded (FFPE) RNA.

Figure 4. Analysis of first-strand cDNA synthesis performed in the presence of both biological and sample prep inhibitors. 500ng of Invitrogen™ Ambion™ 0.5–10kb RNA Ladder (Cat. No. 15623200) was used in a 10μL SuperScript III or SuperScript IV RT reaction with oligo(dT)20 according to the provided protocols. Reverse transcriptases from other vendors were tested using the manufacturers’ recommended protocols. Inhibitors were added to total RNA prior to primer annealing and addition of RT reaction mix. First-strand cDNA was resolved by alkaline gel electrophoresis and stained using Invitrogen™ SYBR™ Gold Nucleic Acid Gel Stain (Cat. No. S11494). NaOH was used to hydrolyze all RNA, resulting in visualization of only cDNA.

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Figure 3. Sensitive cDNA syntheswith a wide range of degraded plant RNA. 1–100ng of degraded Arabidopsis total RNA (RIN: 1–3) was used in a 20µL SuperScript IV RT reaction with random hexamers according to provided protocol. Reverse transcriptases from other vendors were used according to the manufacturers’ recommended protocols. 10% of cDNA in the RT reaction was added to TaqMan Assays for targets indicated in graph titles.

Reason #3: SuperScript IV RT reduces Ct by as much as 8 cyclesSuperScript IV RT has a high affinity for RNA, and due to its high processivity is capable of efficient and sensitive cDNA synthesis of any template. Triplicate qPCR reactions for two Arabidopsis targets were performed; for both targets, SuperScript IV RT resulted in lowest Ct values over all input RNA amount (Figure 3).

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To find out more or to place an order, go to thermofisher.com/ssiv

For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent Technologies, Inc. CO125100 0815

Product Size Cat. No.SuperScript IV Reverse Transcriptase 2000 units 18090010

10,000 units 18090050

4 x 10,000 units 18090200

SuperScript IV First-Strand Synthesis System 50 rxns 18091050

200 rxns 18091200

The SuperScript IV System advantageInvitrogen™ SuperScript™ IV Reverse Transcriptase is available as stand-alone enzyme or in a convenient first-strand synthesis system format. The first-strand synthesis system includes all the necessary components for successful first-strand cDNA synthesis in separate tubes, allowing for maximum optimization of cDNA synthesis enabling successful experiments.

“I tried the SuperScript IV (SSIV) RT with RNA extracted from whole blood (normal sample), plasma (difficult sample), and laser capture samples from skin (difficult sample also). The SSIV RT performed very well in all cases. Better than SuperScript II that I’m usually using, especially for plasma sample. It’s also amazing because I get better results in less time!”

– Researcher, Qgenomics

superscript iv reverse transcriptase— top iv … · with random hexamers according to the...

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