The first-strand cDNA synthesis; RT-PCR.

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The first-strand cDNA synthesis

Following second strand synthesis, transfer contents of 0.5 ml microfuge into a 1.5 ml RNase-free microfuge tube.

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Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM. At the end of the section we present a method for scanning EM (SEM) of worms. In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images ().

Second-Strand cDNA Synthesis Kit-dNTP/dUTP based Applied Biological Materials ..

The muscles used for locomotion in reside in the body wall. In the adult, there are 95 spindle shaped cells divided among four quadrants just underlying a basement membrane, hypodermis and cuticle. In each quadrant, the cells are arranged in interlocking pairs. In these muscle cells, myofilaments form a lattice that is restricted to a narrow zone of ~1.5 microns, just underlying the basement membrane and hypodermis. By polarized light microscopy, obvious striations are seen; bright (“birefringent”) A-bands alternate with dark I-bands; each I-band contains a row of dense bodies, which are the analogs of Z-discs of vertebrate striated muscle (). Because the striations lie at a slightly oblique angle with respect to the long axis of the worm, this muscle is called “obliquely striated”. Polarized light is also useful for evaluating the second largest set of muscles, those in the pharynx. Below we present a protocol for observing muscle using polarized light.

manual NEBNext mRNA Second Strand Synthesis Module E6111S Read ..

Herein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.

Accurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.

NEBNext Second Strand Synthesis Reaction ..

Description: Thermostable M-MLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. M-MLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional . The difference between this to the general M-MLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50℃, it can also keep more than 80% activity even at 55℃.

Currently, clinical laboratories world-wide are using different commercially available kits, based on different primers and enzymes, for the synthesis of cDNA. This can potentially influence the results and consequently the detection of MRD. Yet, no study is available comparing these kits in order to define whether these are suitable for the detection of BCR-ABL1 at the MMR level. In this study, we compare six commercially available cDNA synthesis kits (Table ), for the identification of the most suitable kit for use in a CML MRD assay. For each kit, the manufacturers’ protocols are used with BCR-ABL1/ABL1 samples at 0.32%, 0.032% and 0.0032%, followed by the Europe Against Cancer (EAC) protocol for the detection of BCR-ABL1 and ABL1 gene using the TaqMan® method. The results are analysed and scored according to failure rate, linearity and ABL1 copy number.

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  • Second strand synthesis and amplification ..

    Following second strand synthesis, transfer contents of 0.5 ml microfuge to 1.5 ml RNase-free microfuge tube.

  • Synthesis of the Second-Strand ..

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  • Stranded cDNA Synthesis Kit (Invitrogen) ..

    (Following the protocol outlined in the Invitrogen Double Strand cDNA Synthesis kit - check out the manual here)

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Primer for first-strand cDNA synthesis of ..

Serial measurement of leukemia-specific transcripts is a valuable approach for monitoring individual patients and in some cases for indicating the need to reassess therapy []. Following the high efficacy of the standard CML treatment, the need for an accurate method for monitoring the response is existent. Accurate quantification of BCR-ABL1 transcripts has been proven to be the most sensitive available method with a great prognostic impact and serving for an early assessment of MRD []. Hence, identification of which commercially available kit is more suitable for the synthesis of cDNA is essential, in order to achieve better results at the MMR level. Herein, the six kits tested were classified based on three parameters: a) success rate, b) ABL1 copy number and c) linearity.

Cloned AMV First-Strand cDNA Synthesis Kit - Invitrogen

At first, a success rate analysis was performed, where the reverse transcription enzyme was considered successful if it amplified the BCR from the cDNA. The success rate was calculated based on the number of wells that were successfully amplified divided by the total well number for BCR amplification, with the enzyme with the highest success rate achieving the highest score (with the highest score being 6 and the lowest 1). All kits detected 0.32% BCR-ABL1 copy numbers. Using the SuperScript® III Reverse Transcriptase kit the BCR was successfully amplified from the cDNA at all levels, achieving a score of 6 (18/18). QuantiTect® Reverse Transcription, First Strand cDNA Synthesis kit for RT-PCR and the Transcriptor First Strand cDNA Synthesis kit achieved 15 successful reads at the 0.0032% level, attaining a score of 5 (15/18). AffinityScript Multiple Temperature cDNA Synthesis kit achieved 13 successful reads at the 0.0032% level, attaining a score of 2 (13/18) and the iScript™ Select cDNA Synthesis kit recorded 5 successful reads at the 0.032% level and one successful read at the 0.0032% level (12/18), attaining the lowest score of 1 (Additional file : Table S1).

MessageBOOSTER cDNA Synthesis Kit for qPCR

According to linearity, the ratio of BCR/ABL1 at 0.32% copy numbers is expected to be 10 times more than the ratio of BCR/ABL1 at 0.032% copy numbers. The same also applies for 0.032% versus 0.0032%. In this study, absolute values were calculated using the formulae: ratio of 0.032% − (ratio of 0.32% divided by 10) and ratio of 0.0032% − (ratio of 0.032% divided by 10). Finally, a score of 1 to 6 was given to each kit based on the absolute values observed. Smaller absolute values were given higher scores, with the highest being a score of 6, assigned to the kit with the highest final score after adding the two individual scores. The highest ratios were observed by the SuperScript® III Reverse Transcriptase and AffinityScript Multiple Temperature cDNA Synthesis kit, both achieving a score of 6. Whereas the iScript™ Select cDNA Synthesis kit and the Transcriptor First Strand cDNA Synthesis kit achieved the lowest scores (Additional file : Table S3).

MessageBOOSTER™ cDNA Synthesis Kit for ..

In order to acquire a total score for each kit, the sum of the success rate, ABL1 and linearity scores was observed. Using this strategy, all three parameters were taken into consideration for the evaluation of the different cDNA synthesis kits. Based on our findings, the kit with the highest acquired score was the SuperScript® III Reverse Transcriptase, by Invitrogen, achieving an overall score of 18, followed by the First Strand cDNA Synthesis Kit for RT-PCR, by Roche, with an overall score of 14 (Table ).

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