TSS were identified using the RLM-RACE (Ambion) kit followed by cloning 5’rapid amplification of cDNA ends (5’RACE) products into the pCR4-TOPO vector. Briefly, DNase I- treated RNA was phenol:chloroform:isoamyl alcohol (25:24:1, Invitrogen) extracted and treated with calf alkaline phosphatase (Ambion). Reverse transcription of the RNA adapter-tagged sample was primed using gene specific primers (sequences in Supplemental Table S1 ) using Superscipt III (Invitrogen ) at 50 °C, following manufacturer instructions. A first PCR was carried as described above for 32 cycles using the “outer” Ambion primer with the 1st nested gene-specific primer (Supplemental Table I ). A second (nested) PCR was carried out as above using the “inner” Ambion primer with the 2nd nested gene-specific primer, at one of several annealing/extension temperatures (54 – 68 °C, 60 s) for 40 cycles. Products were purified using a Qiagen spin column and cloned into the pCR4-TOPO vector using the TOPO-TA cloning kit (Invitrogen). Sequencing was carried out by the Northwestern University sequencing facility (Chicago, IL).
Rlm race
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RLM-RACE (RNA Ligase-Mediated Rapid Amplification of cDNA Ends) is a lab equipment product designed for the rapid amplification and identification of the 5' and 3' ends of RNA transcripts. It utilizes a unique RNA ligase-mediated method to generate cDNA from RNA samples, allowing for the efficient capture of full-length transcript sequences.
Automatically generated - may contain errors
Lab products found in correlation
Generacer kit, by Thermo Fisher Scientific (3 mentions)
Calf alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
Spin column, by Qiagen (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
Superscipt 3, by Thermo Fisher Scientific (2 mentions)
Sequencher 4, by Gene Codes (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Pgem t easy vector, by Thermo Fisher Scientific (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
3 race system for rapid amplification of cdna ends, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
12 protocols using rlm race
1
5' RACE Protocol for Transcription Start Sites
2
Cloning and Characterizing Zebrafish sprtn Ortholog
3
TMPRSS2-ERG Fusion Transcript Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Under approved protocol from the WRAMC IRB six cases were identified with TMPRSS2-ERG fusion harboring prostate tumors. Total RNA was isolated from the tumors and were pooled [29 (link)]. From the pool 4.2 μg of total mRNA was subjected to 5’ oligocapping procedure (FirstChoice, RLM-RACE, Ambion, Austin, TX) pairing the 5’-GGCGTTGTAGCTGGGGGTGAG-3’ [11 (link)] with the outer, and 5’- CAATGAATTCGTCTGTACTCCATAGCGTAGGA-3’ with the inner primer. Amplicons were gelpurified and cloned into pUC19 vector and were subjected to DNA sequencing in forward and reverse directions.
4
Determining Full-Length Tph Gene Sequence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the full-length sequence of the S. mediterranea tph gene, EST [17 (link)] and 5’ Rapid Amplification of cDNA Ends (RLM-Race, Ambion, Austin, TX) sequences were assembled using Sequencher 4.7 (Gene Codes, Ann Arbor, MI). These sequence data were aligned to the S. mediterranea genome to determine the structure of the tph gene. While the 5’ and 3’ ends of tph aligned to supercontigs 654 and 6406, respectively, we found 30 nucleotides in exon three that showed no similarity to any sequences in the current S. mediterranea genome assembly. Thus, we used this 30 nucleotide sequence to query S. mediterranea genome sequence reads present in the NCBI trace archive. This analysis identified a single trace (ULF34H07) that included the missing sequence data. Assembly of this trace with supercontigs 654 and 6406 collapsed these sequences into a single contig (Fig 1A ), which represents the only tph gene in this species.
5
Cloning and Characterizing Homologous Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All genes investigated in this study were cloned by methods described previously [37 (link)], and the sequences have been deposited in the GenBank (see Additional file 1 : Table S1 for the accession numbers). In brief, cDNA sequences of homologous genes from S. kowalevskii were retrieved from GenBank and used as queries to search the P. flava embryonic transcriptome database [38 (link)]. Specific primers were designed for PCR (see Additional file 1 : Table S2). RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE, Ambion) was used to obtain the full-length sequences. Total RNA was extracted using TRIzol reagent (Invitrogen) from several developmental stages, including unfertilized eggs, embryos at 12, 17, 25, 32, 43 and 64 hpf, and the Spengel, Agassiz and juvenile stages. The extracted RNA was further purified using an RNeasy Micro kit (Qiagen, Chatsworth, CA, USA). For cloning, the purified RNAs from each stage were reverse transcribed separately to obtain cDNA.
6
5' RACE Protocol for Transcription Start Sites
7
Characterization of miR-22 Genomic Structure
8
Comprehensive Planarian Genome Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly prepared RNA from mature sexual planarians was used for cDNA synthesis (iScript, Bio-Rad) or 5’-RACE (RLM-RACE, Ambion) according to manufacturer instructions. Large overlapping amplicons across the PSCNV genome (primers in S2 Table ) were amplified by standard Phusion® High-Fidelity DNA polymerase reactions, with 65°C primer annealing temperature and 10 min extension steps.
9
Mapping miRNA-Cleaved Transcripts by 5' RACE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the cleaved targets by miR408, 5′ RACE was performed using the GeneRacer kit (full-length, RNA ligase-mediated rapid amplification of 5′ and 3′ cDNA ends, RLM-RACE, Invitrogen®) for mapping the 5′ end of an miR408 target, TC122593, predicted to encode a diphenol oxidase laccase. Briefly, RNA (5 µg) from sugarcane plants was ligated to a 5′ RACE adaptor. Random hexamer primers were then used for cDNA synthesis. PCR amplification of a cDNA fragment containing the cleavage site of the targets was carried out by nested PCR. Primers used in PCR are available in Table S7 . RACE fragments were cloned into a pGEM T-easy vector (Invitrogen®) and sequenced.
10
Isolating DtFLC Gene from Rocket
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Degenerate primers for FLC were designed using aligned sequences for Arabidopsis and Brassica FLC genes and used to PCR amplify DtFLC using D. tenuifolia cDNA as the template. Specific DtFLC primers (Supplementary data Table 1A) were then designed to isolate the remaining sequence using RLM RACE (Invitrogen). 5′ and 3′ UTR primers specific to DtFLC (see supplementary data for sequences) were used to amplify the gene from rocket genomic DNA and cDNA. The gene sequence data has been submitted to the GenBank database under accession number KX148480.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!