Smart race cdna amplification kit

We use the SMART™ RACE cDNA Amplification Kit (Takara, Japan) to amplification the 3′ and 5′ end of linc00423 according to the manufacturer’s protocol. Subcellular fractionation analysis were performed use the Nuclear/Cytosol Fractionation Kit (Pierce, USA).

The coding regions of Unigene16368_All (ZxNAC083) and CL6534.Contig1_All (ZxNAC035) were amplified from seedling total RNA using a SMART RACE cDNA Amplification Kit (TaKaRa Biotechnology, China). The resulting cDNA products were cloned into pDONR™/Zeo using a Gateway® BP reaction (ThermoFisher Scientific, China) then inserted into binary vector pBIB-BASTA-35S-GWR-GFP using a Gateway® LR reaction (ThermoFisher Scientific, China). The resulting constructs were introduced into Agrobacterium tumefaciens strain GV3101 and used to transform wild-type Arabidopsis Col-0 plants by floral dipping (seeds of wild-type Arabidopsis Col-0 were obtained from Ministry of Education Key Laboratory of Cell Activities and Stress Adaptation, Lanzhou, China) [70 (link)]. Transgenic overexpression lines were validated using semi-quantitative RT-PCR to measure ZxNAC083 and ZxNAC035 transcript levels. AtACTIN2 was used as the internal control gene. Primer sequences are provided in Additional file 2: Table S8.

Amplification of 5ʹ and 3ʹ ends of cDNA was performed according to the protocol described in the SMART RACE cDNA Amplification Kit (Takara Bio USA, Inc. Mountain View, CA, USA). Three pairs of gene-specific primers (GSP), one for each hsp70 isoform: hsp70, hsc70-I and hsc70-II, were designed based on the sequences obtained from amplification with degenerate primers (Table 1).
Total RNA of Diamesa tonsa larvae was extracted according to the Trizol protocol (Thermo Fisher Scientific) and the quantity and quality assessed as described above. 1 μg of total RNA was first retrotranscribed with primers supplied in the kit and this first-strand cDNA was used directly in PCR amplification reactions that were achieved using a high-fidelity enzyme (KAPA HiFi DNA Polymerase, Kapa Biosystems), the Universal Primer Short (UPS, supplied by the kit), and gene-specific primer (GSP_F for 3ʹ RACE- cDNA or GSP_R for 5ʹ RACE- cDNA). The PCR was performed with the following cycler protocol: 95°C for 3 min, 25 cycles of 98°C for 30 sec, 68°C for 15 sec and 72°C for 90 sec, and a final extension of 5 min at 72°C.

Rapid amplification of cDNA ends (RACE) was performed using the SMART RACE cDNA Amplification kit (TAKARA). Two primers, GDI-5′ and GDI-3′, each combined with the universal primer A mix, were used to amplify the 5′-cDNA and 3′-cDNA ends of ZmGDIα-hel from the resistant line X178, respectively. The 5′-RACE and 3′-RACE products were cloned into the pEasy-T1 vector for sequencing. The 5′-terminal and 3′-terminal sequences were merged to obtain the full-length ZmGDIα-hel cDNA. With the availability of the ZmGDIα-hel cDNA, primers Race-GDI were designed to amplify the HuangC cDNAs to obtain full-length ZmGDIα cDNAs.

After the leaves were ground into powders, RNA was separately extracted using the Mini BEST Plant RNA Extraction Kit (TaKaRa, Tokyo, Japan) accoridng to the manufacturer’s instructions, genomic DNA (gDNA) was removed by Recombinant DNase I, and all RNA samples were examined with Nanodrop 2000C (Thermo Scientific, Waltham, MA, USA). The full-length cDNA of PlPM19L was isolated via RACE technology using a SMART RACE cDNA Amplification Kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. The specific primers used for 5’/3’ RACE PCR amplification were designed by Primer 5.0 software (Table S1). After being tested via 1% (w/v) agarose gel electrophoresis, the PlPM19L PCR products were cloned into the pClone007 Vector and sequenced.
The amino acid composition, relative molecular weight, isoelectric point, and other physical and chemical properties of PlPM19L were analyzed via Protparam (http://web.expasy.org/protparam) (accessed on 17 September 2022). The protein sequences of the other plants were obtained from the NCBI, and the sequence alignment was analyzed via DNAMAN. A neighbor-joining phylogenetic tree was generated with MEGA 7.0, and bootstrap values were set as 1000 bootstrap replicates [45 (link)].

Total RNAs of the above samples were isolated by TRIzol (Invitrogen, Carlsbad, CA, USA). The RNA quality and quantity were determined by spectrophotometer (Thermo Fisher, Waltham, MA, USA). Then, 1 μg RNA was electrophoresed with a 1.0% agarose gel to analyze the RNA integrity. The remaining RNA sample was frozen at −80 °C. The 5′-RACE-Ready and 3′-RACE-Ready cDNA were obtained by SMART^™ RACE cDNA Amplification Kit (TaKaRa, Otsu, Japan). cDNA was synthesized by the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) [17 (link)]. All operations were performed according to the manufacturer’s instructions.