| Description | EnzymoPure™Q M-MLV Reverse Transcriptase uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand with high fidelity, sensitivity, and specificity. It is a modified, optimized reverse transcriptase particularly suitable for qPCR analysis. The EnzymoPure™Q M-MLV Reverse Transcriptase uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand with high fidelity, sensitivity, and specificity. It is a modified, optimized reverse transcriptase particularly suitable for qPCR analysis. The EnzymoPure™Q M-MLV Reverse Transcriptase contains RNase H enzyme activity that can specifically degrade the RNA strand of an RNA-DNA hybrid, enabling the subsequent synthesis of second-strand cDNA.EnzymoPure™Q M-MLV Reverse Transcriptase, a commonly used high-quality reverse transcriptase, is widely used in the synthesis of first-strand cDNA using total RNA or mRNA templates, and is particularly suitable for qPCR and one-step qRT-PCR analysis. This product is also applicable for conventional PCR, synthesis of second-strand cDNA and construction of cDNA library, and cloning of target genes. It can also be used for DNA probe labeling with fluorescence, biotin, digoxin, or isotope through reverse transcription reaction, or for RNA studies by primer extension.Please refer to Figure 1 for the qPCR analysis of the GADPH gene in HEK293T cells using the cDNA templates obtained by the EnzymoPure™Q M-MLV reverse transcriptase.Figure 1Source：Recombinant reverse transcriptase expressed in E. coli.Definition of enzyme activity unit: One unit is defined as the amount of enzyme that incorporates 1 nmol of dTTP into acid-precipitable material in 10min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. Reaction system: 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP, and 0.4mM polyA•oligo(dT)12-18.Purity: This product is free from DNA endonuclease, exonuclease, phosphatase, and RNase, and can meet the requirements of conventional reverse transcription.Storage buffer: 20mM Tris-HCl (pH7.5), 150mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40, and 50%(v/v) glycerolInactivation or inhibition：RT Q M-MLV Reverse Transcriptase can be inactivated by incubation at 80℃ for 10 minutes, or inhibited by EDTA and EGTA chelators, inorganic phosphates or pyrophosphates, and polyamine.The concentration of M-MLV is 200U/µl. When 20µl of reverse transcription reaction volume is used, D7188S, D7188M, and D7188L are sufficient for 50, 250, and 1000 reactions, respectively. provides a variety of reverse transcriptase. To select the best one for your needs, please refer to the following webpage:http://www.aladdin-e.com/support/reversetranscriptase.htmPrecautions：Please refer to the instructions for reverse transcription of RNAs with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. First-strand cDNA synthesisa. Set up the following reaction on ice or at room temperature. RNase Inhibitor and dNTP mix can be purchased from SYBR Green qPCR Mix (2X), SYBR Green qPCR Mix (2X, Low ROX), and SYBR Green qPCR Mix (2X, High ROX) are for conventional qPCR analysis with SYBR Green fluorescent dye. Probe qPCR Mix (2X), Probe qPCR Mix (2X, Low ROX), and Probe qPCR Mix (2X, High ROX) are for qPCR analysis with Taqman Probe.b. Perform qPCR analysis following the instructions of the corresponding qPCR kit.3. For other applications such as primer extension and probe labeling, please refer to references related to M-MLV reverse transcriptase.FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different sizes is even lower. 2. No specific product is amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. If reference genes can be amplified, but not the target gene, it indicates primers of the target gene are not well designed. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, spermidine, etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol or Trizol produced by can fully meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I is used to remove the residual DNA in the RNA sample prior to reverse transcription and is subjected to heat-inactivation, EDTA should be added to the RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. An inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well-designed... Read More | Inquire | Inquire | Inquire | Vabicaserin hydrochloride is a 5-hydroxytryptamine 2C ( 5-HT 2C ) receptor -selective agonist with an EC 50 of 8 nM.In VitroVabicaserin displaces 125 I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT 2C receptor sites in Chinese hamster ovary cell membranes with a K i value of 3 nM and Vabicaserin hydrochloride is a 5-hydroxytryptamine 2C ( 5-HT 2C ) receptor -selective agonist with an EC 50 of 8 nM.In VitroVabicaserin displaces 125 I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT 2C receptor sites in Chinese hamster ovary cell membranes with a K i value of 3 nM and is >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT 2B receptor subtype using [ 3 H]5HT is 14 nM. Vabicaserin is a potent and full agonist (EC 50, 8 nM; E max, 100%) in stimulating 5-HT 2C receptor-coupled calcium mobilization and exhibits 5-HT 2A receptor antagonism and 5-HT 2B antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. Vabicaserin exhibits lower affinity at the 5-HT 2C antagonist binding site (22 nM) labeled with [ 3 H]mesulergine. Additional binding studies indicate that Vabicaserin possesses affinity for the 5-HT 2B and 5-HT 1A receptors with K i values of 14 and 112 nM, respectively. MCE has not independently confirmed the accuracy of these methods. They are for reference only.In VivoAfter a single oral dose of [ 14 C]Vabicaserin at 50, 5, and 15 mg/kg, unchanged drug represents less than 19, 20, and 35% of total plasma radioactivity at all the time points examined in mice, rats, and dogs, respectively. The carbamoyl glucuronide (CG) represents approximately 7 to 36% of plasma radioactivity in mice and 2 to 28% of plasma radioactivity in dogs but is not detected in rat plasma after the single [ 14 C]Vabicaserin dose. However, the CG is observed in rat plasma after multiple-dose administration of Vabicaserin at higher doses, and the CG is approximately 20 times less than Vabicaserin based on steady-state AUC 0-24 values. The estimated plasma AUC 0-24 ratios of CG to the parent drug are 1.5 and 1.7 in mice and dogs after the single [ 14 C]Vabicaserin dose, respectively. The plasma AUC 0-24 ratios for the CG to Vabicaserin at steady state with doses used for safety assessment are less for mice (0.2-0.6) and slightly higher for dogs (1.8-4.0) compared with the single dose values. The CG is detected in dog urine in similar amounts to the parent drug, although it is not detected in mouse or rat urine after the single [ 14 C]Vabicaserin dose. Radioactivity in a 0- to 24-h bile collection from rats receiving a 5 mg/kg [ 14 C]Vabicaserin dose accounts for 19 and 24% of the administered dose in males and females, respectively. Although the CG is not detected in urine or feces of rats after a single oral administration, it represents an average of up to 30% of biliary radioactivity in male rats and 15% in female rats. In monkeys after a single oral 25-mg/kg dose of Vabicaserin, the plasma concentrations of the CG exceeded those of Vabicaserin at all the time points (2-24 h) postdose, although the amount of CG relative to Vabicaserin decreased by 24 h postdose, with ratios of 17.5 at 2 h and 1.7 at 24 h. The CG to Vabicaserin AUC 0-24 ratio of 12:1 indicates that the CG is a major metabolite in monkeys. MCE has not independently confirmed the accuracy of these methods. They are for reference only.Animal administrationMice and Rats For metabolism studies in mice, rats, and dogs, radiolabeled doses are used. Male and female CD-1 mice and Sprague-Dawley rats are used. The dose vehicle for mice and rats contained 2% (w/w) Tween 80 and 0.5% methylcellulose in water. Nonfasted male and female mice weighing from 27.8 to 33.8 g at the time of dosing are given a single 50-mg/kg (∼300 µCi/kg) dose of Vabicaserin at a volume of 20 mL/kg via intragastric gavage. Mice are kept in metabolic cages in groups of five. Nonfasted male rats weighing from 318 to 345 g and female rats weighing from 227 to 255 g at the time of dosing are given a single 5-mg/kg (∼300 µCi/kg) dose of Vabicaserin at a volume of 2.5 mL/kg via intragastric gavage. Four bile duct-cannulated male rats weighing from 387 to 411 g and four bile duct-cannulated female rats weighing from 291 to 325 g at the time of dosing are nonfasted and are given a single 5-mg/kg (323 µCi/kg) dose of Vabicaserin at a volume of 5.0 mL/kg via intragastric gavage. Rats are kept individually in metabolism cages. Dogs Four male beagle dogs, weighing from 7.6 to 9.8 kg at the time of dosing, are from an in-house colony. Approximately 11 mg of [ 14 C]Vabicaserin hydrochloride and 940 mg of nonlabeled Vabicaserin hydrochloride are dissolved in methanol and then evaporated under a nitrogen stream to dryness. Capsules (number 2) are filled with accurate amounts (126.7-138.1 mg) of the mixed drug substance according to animal weights to give a dosage of 15 mg/kg (39 µCi/kg). The filled gelatin capsules are then enteric-coated manually. Each dog is given one enteric-coated capsule containing [ 14 C]Vabicaserin as the hydrochloride salt. Animals are fed 2 h before dosing and are housed individually in metabolic cages. Monkey Four male cynomolgus monkeys, weighing from 5.4 to 9.6 kg at the time of dosing, are from an in-house colony. Nonfasted monkeys are given a single 25-mg/kg dose of nonradiolabeled Vabicaserin at a volume of 2 mL/kg via intragastric gavage. The vehicle is the same as used in mice and rats. Animals are housed individually in metabolic cages. aladdin has not independently confirmed the accuracy of these methods. They are for reference only.IC50& Target:5-HT 2C Receptor 8 nM (EC 50 )... Read More |