Design
A molecular biology experiment.
Time and setting
Experiments were performed from September 2010 to December 2010 in the Department of Histology and Embryology, Southern Medical University, China.
Materials
A pathogen-free pregnant Sprague-Dawley rat was provided by the Animal Experimental Center of Southern Medical University (Guangzhou, China; license No. SCXK (Yue) 2006-0015). The animal was housed at 22 ± 3°C, with 55 ± 3% relative humidity and a 12-hour light/dark cycle, allowing free access to food and water. Animal procedures were in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[31].
Methods
Design of nested reverse transcription-PCR primers
Two pairs of nested PCR primers for the N-terminal product of sonic hedgehog were designed using Primer Premier Software (Version 5.0, Canada), using the sequence of rat sonic hedgehog published in GenBank (NM_017221.1) as reference. Primer pairs were BLASTed (NCBI) to avoid annealing to non-specific sequences during amplification. PCR products using these primers were expected to be 525 bp long. Primers were diluted to a working concentration of 10 μM. Primer synthesis was conducted by Invitrogen Corporation (Shanghai, China). The primers used are as follows:
Extraction and reverse transcription of total RNA
A pregnant Sprague-Dawley rat was anesthetized with 10% chloral hydrate (Shanghai Aladdin Reagent Co., Ltd., China); 0.64 g of notochord was excised from fetuses using Rnase-free materials and reagents. Total RNA was isolated from the notochord using an RNAiso Plus kit (Takara, Guangzhou, Guangdong Province, China) according to the manufacturer’s instructions. The RNA was run out on a 1% agarose gel to determine the quality of extraction. RNA yields were measured at a wavelength of 260 nm on a Bio-Rad Smartspect 3000 spectrophotometer (Bio-rad, Guangzhou, Guangdong Province, China). The concentration of total RNA in solution was then calculated using the formula: absorbance units × 71/40 = μg of RNA.
The cDNA was synthesized using a RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Guangzhou, Guangdong Province, China), 5 μL of RNA were heated at 65°C for 5 minutes with 1 μL of OligodT primer (Shang Aladdin Reagent Co. Ltd., China) and this mixture was placed on ice for 3 minutes after heating. 2 μL of dNTP mix (dATP, dCTP, dGTP, dTTP), 4 μL of 5 × reaction buffer, 1 μL of RiboLock™ RNase Inhibitor and 1 μL of RevertAid™ Reverse Transcriptase were added to the solution. This mix was heated at 42°C for 60 minutes. The reaction was terminated by heating at 70°C for 5 minutes, and the products were diluted 10-fold in RNAse-free water and stored at –70°C.
cDNA amplification by nested PCR
PCR was performed using primers for β-actin or rat sonic hedgehog to amplify the cDNA with DreamTaq™ Green PCR Master Mix (Fermentas, Guangzhou, Guangdong Province, China). Reagents were added on ice into 200 μL thin PCR tubes. The final reaction mix consisted of 2 μL cDNA, 25 μL Dream Taq Green PCR master mix, 1.5 μL forward and reverse primers, and was made up to 50 μL with PCR quality water. The reaction tubes were then placed into a thermal light-cycler (Biometra, Guangzhou, Guangdong Province, China) which was programmed for the first round as follows: 95°C for 3 minutes, then 25 cycles at 95°C for 30 seconds, 50.2°C for 1 minute, and 72°C for 1 minute. After the cycles were completed, the reaction was left at 72°C for 15 minutes before being cooled to 4°C. The PCR product from this first round was diluted 5-fold in ddH2O. The second reaction mix consisted of 2 μL diluted PCR product from the first round, 25 μL Dream Taq Green PCR master mix, 1.5 μL forward and reverse primers, and was made up to 50 μL with PCR quality water. The second round was programmed as follows: 95°C for 3 minutes, then 35 cycles at 95°C for 30 seconds, 69.8°C for 1 minute, and 72°C for 1 minute. After the cycles were completed, the reaction was left at 72°C for 15 minutes before being cooled to 4°C. Products were run out on a 1% agarose gel to determine size and specificity.
Purification of the PCR product
The PCR product from the second round was purified using a GeneJET PCR purification kit (Fermentas, Guangzhou, Guangdong Province, China). First, the binding buffer (1:1) was added to the second round PCR mix, and mixed thoroughly. Then we transferred this mix solution to the GeneJET™ purification column and centrifuged the column at 13 000 × g for 60 seconds before discarding the flow-through. We then added 700 μL of wash buffer to the GeneJET™ purification column and centrifuged the column at 13 000 × g for 60 seconds. After the flow-through was discarded, the purification column was placed back into the collection tube. The empty GeneJET™ purification column was centrifuged for an additional 1 minute to completely remove any residual wash buffer. Then we transferred the GeneJET™ purification column to a clean 1.5 mL microcentrifuge tube, after which 50 μL of elution buffer was added to the center of the GeneJET™ purification column membrane and centrifuged for 1 minute. Finally, we discarded the GeneJET™ purification column and stored the purified DNA at –20°C.
Bioinformatical analysis of the N-terminal product of Sonic hedgehog with online tools
The nucleotide and amino acid sequences of the N-terminal product of sonic hedgehog were analyzed as previously described[32]. The secondary structure of the N-terminal fragment of sonic hedgehog was predicted with the Jpred online tool (http://www.compbio.dundee. ac.uk/www-jpred/), and the tertiary structure of the N-terminal fragment of sonic hedgehog was predicted with Phyre (http://www.sbg.bio.ic.ac.uk/~phyre/).
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