SuperScript IV Reverse Transcriptase

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Bathsheba Wilkins
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1 WHIE AER Reverse ranscriptase Reverse ranscriptase Abstract Surveys and interviews conducted over a three-year period revealed that researchers are not satisfied with their current reverse transcriptases (Rs), and are performing reactions with increasingly difficult samples such as poorly purified RA that contains inhibitors, RA from plants, formalin-fixed, paraffin-embedded (FFE) samples that are typically degraded, and unpurified RA (direct reverse transcription). o meet this performance gap, we developed Invitrogen SuperScript IV R. Based on internal experimental evidence, it is the most robust R compared to other enzymes when used with difficult RA samples. Characterization of R was performed in the context of imperfect situations where users do not have ideal RA samples. Using a variety of stringent assays, this paper demonstrates that SuperScript IV R possesses superior performance in the presence of a variety of inhibitors, such as alcohols, salts, detergents, heparin, hematin, bile salts, and formalin, typically found in sample preparation reagents, cell lines, blood, feces, and FFE samples. In our experiments, R maintained the highest sensitivity with degraded RA (RA integrity number (RI): 1 3) and unpurified RA samples. Furthermore, this enzyme retains the lowest variability with different amounts of input RA. Finally, R is found to be the most thermostable (1% activity up to 56 C and 9% activity at 6 C) and processive (up to 9 kb in just 1 min) R. o demonstrate even greater value, the attributes of R mentioned above were benchmarked against other leading Rs. Introduction Rs are a class of enzymes that synthesize cda from an RA template. raditionally, scientists used Rs to clone genes and study expressed genes from whole organisms or cells. However, the scientific landscape in gene sequencing has drastically changed in recent times to include single-cell analysis, sample preparation free analysis, and next-generation sequencing, requiring new Rs with single-copy sensitivity, speed, resilience to difficult samples, and thermoreactivity. o meet these demands, we have engineered a new R. R is the newest member of the SuperScript family of Rs, which is known for its quality and reliability in cda synthesis. Functions of Rs are described by several attributes processivity, sensitivity, reproducibility, and yield. While many Rs meet users expectations in these attributes with high-quality RA samples, in cases where inhibitors are present, RAs are degraded, and sample preparation is difficult, Rs from other suppliers fall short of expectations while R excels. Materials and methods RA purification Wheat germ and flax seeds purchased from a grocery store were ground to a fine powder in liquid nitrogen. RA was extracted and purified using the Invitrogen urelink lant RA Reagent (hermo Fisher Scientific, Cat. o ) and its accompanying protocol. otal RA was quantitated using the hermo Scientific anodrop instrument and treated with Dase I (hermo Fisher Scientific, Cat. o ). RA quality was assessed using the Bio-Rad Experion Automated Electrophoresis System and agarose gel electrophoresis with ethidium bromide staining. Degraded RA preparation HeLa RA (hermo Fisher Scientific, Cat. o. AM7852) and Arabidopsis RA (BioChain, Cat. o. R163431) were degraded to RI 1 3 by addition of MgCl 2 to a final concentration of 1 mm and heated to 95 C for 15 min.

2 Unpurified RA (direct reverse transcription) samples elleted HeLa cells, Arabidopsis tissue, wheat germ tissue, and flax seed tissue were ground to a fine powder in liquid nitrogen. he powder was transferred to a microfuge tube and ris-eda (E) was added, vortexed, and centrifuged to pellet debris. Resulting clarified supernatant was transferred into a fresh microfuge tube. rior to reverse transcription, EDA and D were added to the supernatant to a final concentration of 1 mm and 5 mm, respectively, and heated to 95 C for 1 min. Commercial RA samples Commercial RA used included Invitrogen Cervical Adenocarcinoma (HeLa-S3) otal RA (hermo Fisher Scientific, Cat. o. AM7852), Arabidopsis otal RA (BioChain, Cat. o. R163431), Rat Brain otal RA (Clontech, Cat. o ),.5 1 kb RA Ladder (hermo Fisher Scientific, Cat. o ), and Invitrogen Millennium RA Markers (hermo Fisher Scientific, Cat. o. AM715). cda synthesis protocol he example procedure below (Schematic 1) shows appropriate volumes for a single, 2 μl reverse transcription reaction. For multiple reactions, prepare a master mix of components common to all reactions Steps rocedure rocedure details Anneal primer to template RA repare R reaction mix Combine annealed RA and R reaction mix Incubate reactions Optional: remove RA CR amplification a. Combine the following components in a reaction tube. ote: Consider the volumes for all components listed in steps 1 and 2 to determine the correct amount of water required to reach your final reaction volume. Component Volume 5 µm oligo (d) 2 primer, 5 µm random hexamers, or 2 µm gene-specific reverse primer 1 µl 1 mm d mix (1 mm each) 1 µl emplate RA (1 pg 5 µg total RA or 1 pg 5 ng mra) up to 11 µl DEC-treated or nuclease-free water to 13 µl b. Mix and briefly centrifuge the components. c. Heat the RA-primer mix at 65 C for 5 min., and then incubate on ice for at least 1 min. a. Vortex and briefly centrifuge the 5X R buffer. b. Combine the following components in a reaction tube. Component Volume 5X R buffer 4 µl 1 mm D 1 µl RaseOU Recombinant Rase Inhibitor 1 µl Reverse transcriptase (2 U/µL) 1 µl c. Cap the tube, mix, and then briefly centrifuge the contents. Add R reaction mix to the annealed RA. a. If using random hexamer, incubate the combined reaction mixture at 23 C for 1 min., and then proceed to step b. If using oligo (d) 2 or gene-specific primer, procced directly to step b. b. Incubate the combined reaction mixture at 5 55 C for 1 min. c. Inactivate the reaction by incubating it at 8 C for 1 min. ote: Amplification of some CR targets (>1 kb) may require removal of RA. o remove RA, add 1 µg E. coli Rase H, and incubate at 37 C for 2 min. Use your R reaction immediately for CR amplification or store it at 2 C. ote: As a recommended starting point for CR, reverse transcription reaction (cda) should compose 1% of the total reaction volume. Schematic 1. Steps for first-strand cda synthesis using Invitrogen SuperScript IV First- Strand cda Synthesis System. to minimize pipetting error, then dispense appropriate volumes into each reaction tube prior to adding annealed template RA and primers. qcr he reverse transcription reaction contributed up to 1% of the total qcr reaction volume. Applied Biosystems aqman Assays for the gene targets are indicated in the figures. EXRESS qcr SuperMix (hermo Fisher Scientific, Cat. o K) and the Applied Biosystems ViiA 7 Real-ime CR System (hermo Fisher Scientific, Cat. o ) were used for qcr. C t values were normalized to R using the equation: ormalized Y values = 2^(C t C t competitor). Endpoint CR he reverse transcription reaction contributed up to 1% of the total CR reaction volume. Invitrogen latinum aq DA olymerase High Fidelity and its accompanying protocol were used. 1 μl of CR reaction was resolved using agarose gel electrophoresis and visualized by ethidium bromide staining. Activity assay for thermostability Reverse transcriptases were preincubated at the indicated temperatures for an indicated amount of time in 1X reaction buffer and 1 ng/µl Invitrogen Ultraure Calf hymus DA Solution (hermo Fisher Scientific, Cat. o ). Following preincubation, enzymes were added to a 1X polymerization mix containing 1X reaction buffer, 2 mm oligo(d) 16,.2 µg poly(ra) (GE Healthcare, Cat. o ), 2 mm d, and 1X EvaGreen dye (Biotium, Cat. o. 31). Extension was performed for 1 min at room temperature. Fluorescence reading was recorded using the SpectraMAX Gemini EM plate reader (Molecular Devices) with an excitation of 49 nm and emission of 52 nm. ercent activity remaining after heat treatment was determined by normalizing to the fluorescence reading without heat treatment that is set to 1%.

3 First-strand cda synthesis for thermostability Reverse transcription was performed as described above using oligo(d) 2 and 5 ng Millennium RA Markers, with the exception of reaction temperature that ranged from 5 to 65 C. Invitrogen SuperScript III R reactions were performed according to the accompanying protocol with the exception of reaction temperature that ranged from 5 to 65 C with a reaction time of 5 min. First-strand cda was resolved by alkaline gel electrophoresis and cda was stained using Invitrogen SY Gold ucleic Acid Gel Stain (hermo Fisher Scientific, Cat. o. S11494). Sodium hydroxide hydrolyzed all RA, resulting in only visualization of cda. Each cda band was measured using the otallab image analysis module and volumes were summed up for each reaction temperature. ercent activity was calculated from the ratio of total volume at each reaction temperature to the total volume at 5 C. Results R-qCR performance using R and Rs from other suppliers in the presence of inhibitors race amounts of reagents used during RA isolation can cause problems during reverse transcription. For example, some reagents used to lyse cells, such as SoluLyse and BugBuster reagents, contain detergents. RIzol reagent, used to extract RA from cells and tissues, contains phenol. Salts such as guanidinium chloride, guanidinium isothiocyanate, ammonium acetate, and lithium chloride are used in multiple steps during RA isolation and precipitation. FFE samples may still contain formalin and paraffin. Inhibitors may also be inherent in the biological sample source such as hematin, a drug found in blood, bile salt, found in blood and feces, and humic acid, found in soil and thus, on plants. o test how chemical compounds affect reverse transcription efficiency, various inhibitors were added to total HeLa RA prior to the oligo(d) 2 annealing step. Concentrations indicated in Figure 1 are the final concentrations of inhibitors in complete reverse transcription reactions. Reverse transcription with R, R, and six other Rs (,,,,, and ) from other suppliers, followed by qcr, revealed that R had the most consistent results with all of the inhibitors tested (Figure 1). R functions exceedingly well, better than all other tested enzymes in the presence of ethanol, SoluLyse reagent, salts, hematin, and humic acid. he enzyme from supplier gave ~5-fold more product than R. he reactivity of R is almost equivalent to that of supplier enzyme in BugBuster reagent. Supplier and enzymes functioned better than in formalin. Since the majority of reverse transcriptases functioned more uniformly in paraffin, the wax component in FFE samples caused little to no inhibition in reverse transcription. In summary, SuperScript IV R demonstrated the most consistent performance in the presence of a variety of inhibitors. ormalized R efficiency % Ethanol 2% Isopropanol 1% RIzol 4% SoluLyse 4% BugBuster % Formalin 2% araffin 1.2 ormalized R efficiency M guanidinium chloride.1 M guanidinium isothiocyanate.2 M ammonium acetate.2 M lithium chloride 4 µm hematin 5 ng/µl humic acid Figure 1. R-qCR performance with R and Rs from other suppliers in the presence of inhibitors.

First-strand cda synthesis performance using R and Rs from other suppliers in the presence of inhibitors In addition to studying the effect of inhibitors on RqCR, direct analysis of R activity in the

4 First-strand cda synthesis performance using R and Rs from other suppliers in the presence of inhibitors In addition to studying the effect of inhibitors on RqCR, direct analysis of R activity in the presence of inhibitors was also performed. R-qCR may hide the actual performance of Rs because targets are usually less than 2 bp and thus, much easier to reverse-transcribe. herefore, analysis of first-strand cda synthesis using RA targets of different sizes gives a more accurate representation of R performance. First-strand cdas resulting from reverse transcription of a.5 1 kb RA ladder were resolved by alkaline gel electrophoresis that degrades RA. Single-stranded cda was visualized by staining with SY Gold ucleic Acid Gel Stain. Contrary to the results observed with R-qCR of small targets, the reverse transcription of longer targets revealed that the enzymes are more susceptible to inhibitors in general (Figure 2). In the presence of isopropanol, the enzyme from supplier does not make any cdas of size.5 kb or below, while R can still synthesize up to 1 kb. In BugBuster reagent, R can synthesize up to 8 kb, while supplier enzyme makes very little cda and stops after ~1.5 kb. he same effect was observed for formalin, where supplier and enzymes had trouble with cda yield and length, while SuperScript IV R retained most of its activity. Other inhibitors tested by direct cda analysis encompassed inhibitors inherent in biological samples such as heparin found in animal blood, tissue, and cells and bile salts found in blood and feces. Using analysis of just first-strand cda, R exhibited superior performance compared to reverse transcriptases from all other suppliers. he exception was with bile salts, where R also retained most of its activity. ositive control experiments where no inhibitors were added to reverse transcription revealed that,,, and enzymes from other suppliers cannot synthesize targets greater than 2 kb even when conditions are ideal. 1% isopropanol 5% BugBuster.25% formalin.1 U/µL heparin.5% bile salts o inhibitors kb Figure 2. First-strand cda synthesis performance using R and Rs from other suppliers.

erformance of R and Rs from other suppliers with degraded RA samples Sensitivity, or the ability of Rs to generate cda from very little input RA, is an important attribute to this class of enzyme.

5 erformance of R and Rs from other suppliers with degraded RA samples Sensitivity, or the ability of Rs to generate cda from very little input RA, is an important attribute to this class of enzyme. Furthermore, researchers are working with increasingly difficult sample sources where RA becomes degraded during the RA purification process, resulting in even lower yields of fulllength transcripts. R sensitivity was therefore evaluated in the context of degraded RA from HeLa cells, Arabidopsis tissue, wheat germ tissue, and flax seed tissue (Figure 3). RI values ranged between 1 and 3, in contrast to high-quality, intact RA with an RI greater than 8. ine targets were evaluated by qcr and in every case, R was more sensitive than all other enzymes tested. erformance of R and Rs from other suppliers with unpurified RA samples he data presented earlier demonstrates that R is the most robust and sensitive R in the presence of inhibitors and degraded RA. However, researchers face a combination of problems with their samples. o challenge Rs with a real world scenario, direct reverse transcription was performed with unpurified RA samples. Ground 293 cells, Arabidopsis tissue, and 25/28S 18S 5S RI 1 3 RI > 8 HeLa Arabipdopsis Wheat germ Flax seeds ormalized R sensitivity FRC OLE-1 18S rra Gin synthetase WRKY F 7 Human cell line RA Arabidopsis Flax seeds Figure 3. erformance of R and Rs from other suppliers with degraded RA samples. ormalized R sensitivity Human cells Figure 4. erformance of R and Rs from other suppliers with unpurified RA samples. GADH Wheat germ EF1 EIF5A FRC OLE-1 18S rra Gin synthetase WRKY F 7 GADH EF1A Arabidopsis Wheat germ wheat germ tissue were mixed with E to dissolve RA, which was then added directly to reverse transcription reactions. hus, the input samples have very low copies of transcripts and a variety of inhibitors. For seven qcr targets in 293 cells, Arabidopsis tissue, and wheat germ tissue, R is the most sensitive (Figure 4). Most Rs performed well for GADH in wheat germ. onetheless, R is the most consistently robust enzyme for direct reverse transcription. EIF1

6 Sensitivity and variability of R and Rs from other suppliers A more thorough investigation of R sensitivity and variability was performed in the context of degraded RA purified from Arabidopsis (RI: 1 3). 1, 1, and 1 ng of degraded total RA was used in reverse transcription reactions. riplicate reverse transcription reactions were performed for each input RA amount. riplicate qcr reactions for two Arabidopsis targets were performed for each reverse transcription reaction. For both targets, R resulted in the lowest C t values over all input RA amounts (Figure 5). A plot of standard deviations for all input RA amounts revealed that not only did R have the lowest standard deviation, but also resulted in the least amount of variation over all three input RA amounts, compared to other Rs. Ct Sensitivity Degraded Arabidopsis RA target: Gin synthetase log 1 [degraded RA] (ng) Degraded Arabidopsis RA target: WRKY F 7 Standard deviation Variability Degraded Arabidopsis RA target: Gin synthetase Reverse transcriptases Degraded Arabidopsis RA target: WRKY F 7 Input RA amount 1 ng 1 ng 1 ng Ct log 1 [degraded RA] (ng) Standard deviation Reverse transcriptases Input RA amount 1 ng 1 ng 1 ng Figure 5. Sensitivity and variability of R and Rs from other suppliers with degraded plant RA.

rocessivity study of R and Rs from other suppliers rocessivity is the ability of a polymerase to perform consecutive nucleotide additions without releasing the RA template.

7 rocessivity study of R and Rs from other suppliers rocessivity is the ability of a polymerase to perform consecutive nucleotide additions without releasing the RA template. he more processive an enzyme, the longer the cda that can be synthesized and the faster the enzyme is in making full-length cda. he R speed and produced cda length were assessed using a.5 9 kb RA ladder. R reaction time was 1 min. Only R synthesized up to 9 kb cda (Figure 6). R can synthesize up to 5 kb, while the rest of the enzymes failed to synthesize cda up to 3 kb, indicating that R is the most processive R. kb hermostability study of R and Rs from other suppliers hermostability of the Rs was evaluated by preincubating at 5 C from to 9 min. Following preincubation, polymerization activity was measured using a fluorescence activity assay. Only and IV Rs remained active at 5 C for a sustained period of time (Figure 7). he thermostability of and IV Rs was more stringently evaluated from 5 C to 65 C by measuring first-strand cda synthesis of a.5 9 kb RA ladder. he cda yield and length dropped drastically with hermostability 5 C.5 Figure 6. rocessivity study of R and Rs from other suppliers. temperatures slightly above 5 C for R. However, R sustained 1% activity up to 56.4 C and 7% activity up to 65 C. he ability of R to function at higher temperatures enables the reverse transcription of RA targets with strong secondary structure. hermostability 55 C Activity (%) reincubation time (min) Activity (%) reincubation time (min) R activity from 5 65 C Activity (%) emperature ( C) Figure 7. hermostability study of R and Rs from other suppliers.

8 cda length study with R and Rs from other suppliers Although most processed RAs are around 3 kb, there are genes that exceed this size. o evaluate the performance of Rs with longer transcripts, reverse transcription followed by endpoint CR was performed for a 12.3 kb in vitro transcript. he reverse transcription protocol was modified to include a gene-specific R primer. Reverse transcription reaction incubation was 3 min at recommended reaction temperatures, 15 min at 5 C above the recommended reaction temperature, and 15 min at 1 C above the recommended reaction temperature for all enzymes tested to accommodate for secondary structure and large transcript size. Subsequent CR resulted in a 12.3 kb product only in the R reaction, while all other Rs produced smears and smaller products (Figure 8). kb λ DA/Hind III Figure 8. cda length study using R and Rs from other suppliers. Conclusion Scientific advancement requires researchers to continually undertake new challenges and obtain results more rapidly. We strive to meet the needs of the scientific community by continuously working on improving reagents and tools for current and future research. R is such an example. R enables scientists to quickly progress their research by providing sensitive and reliable RA analysis from difficult sample sources that contain low copies of target RA, degraded RA, and inhibitors. R raises the bar by enabling scientists to analyze RA without purification. Ordering information roduct uantity Cat. o. Reverse ranscriptase First-Strand Synthesis System VILO Master Mix VILO Master Mix with ezdase Enzyme 2, units , units x 1, units reactions reactions reactions reactions reactions reactions Find out more at thermofisher.com/ssiv For Research Use Only. ot for use in diagnostic procedures. 217 hermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of hermo Fisher Scientific and its subsidiaries unless otherwise specified. aqman is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Experion is a trademark of Bio-Rad Laboratories, Inc. EvaGreen is a trademark of Biotium, Inc. SpectraMAX is a trademark of Molecular Devices, LLC. RIzol is a trademark of Molecular Research Center, Inc. BugBuster is a trademark of Merck, KGAA. SoluLyse is a trademark of Genlantis, Inc. otallab is a trademark of otallab Ltd. COL

SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes

WHITE PAPER SuperScript IV Reverse Transcriptase SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes Abstract Reverse transcriptases (RTs) from avian myeloblastosis virus