Novel LncRNA choice
novel LncRNA selection principles:
1. not overlap with other genes selected from exon lncRNA, otherwise, RT-qPCR results on mixing the expression of two genes.
2. lncRNA best choice between genes, lincRNA i.e., do not overlap with other genes.
3. Second selection gene intron located in other regions lncRNA.
4. Again, if a portion of exon lncRNA overlaps with other genes, the other part does not overlap, we chose not to overlap the portion.
Novel LncRNA primer design
Here we selected novel LncRNA is LNC_000332, LNC_000332 the fasta sequence is as follows:
>LNC_000332:
TGGCAGCTTCAAGAACGAGTGATTTAAGAACAGCCCTCCCATCTTAGCAATGTCCCGGGGTGGCTGGAGCCACGGTCACTTCTTGGTCCTGGTCCAGAACTGTCGGTAGCGCTCCACATGCAAGTCATCACTGAGCTCCTGCTCGTACTCCTTCCTCGACTGGAGCTGAGCCTCACGCTTCCGCAGCCTGGCCCGGCGCTCCTCAGGAGACAGGGTGTCCAGATCTATGGGCATCGCCAGCATGCGCCGTGGCTCCCTGTAGCGGATGTGGATGGTGGAGCCATCCTGCTTCACCAGCAGCACGGGGTAGAGTCGTGCATAAGCCTGGCGGTGCACACGAGTGAGTGAGGCCCTGCTGCTGTCAGCTCGCCAGGAGGATGTGTGCAGGCGGCGGAGTGCAGGTCCGGTGGCCTTCACGGTGCTCTGCCTCAGCCGGCCAAGCAGGCTGCCCACGGCCGCCATTCCTCCTTACAGCCCCAGTGGCACGTGGAGGTGGTCAGTACAGGCCCTGGCGTGCAACTGCGTTGGGTGCTGCTGCGCTGCAGCCCACGACGTCACTGGCAGGCGCTGCGTCGCGCGGTATGACGTGTTCCATGGC
A DNA sequence corresponding to the position of the chromosomal location LncRNA
LNC_000332 chromosomal location: CHR1: 228106685-228109338 , in the minus strand of the chromosome, we obtain the DNA sequence corresponding to the position by UCSC. UCSC's official website http://genome.ucsc.edu
然后选择Genomes,进入后点击View,然后选择DNA
然后在上面的position输入基因的坐标，由于我们的坐标在DNA的负链,所以这里也要勾选Reverse complement,最后点击get DNA,就得到我们想要的DNA序列，下图为得到的DNA序列
通过AnnoLnc获取novel LncRNA的外显子坐标
AnnoLnc是北大生物信息中心开发出的一款分析novel LncRNA的网页工具，网址：http://annolnc.cbi.pku.edu.cn/index.jsp
我们输入LNC_000332的fasta序列，然后点击GO
信息很多，这里只截取一部分，可以看到LNC_000332包含4个外显子区域，不过这是hg19的染色体位置，我们需要用网页版的LiftOver来进行hg19和hg38的坐标转换，网址：http://genome.ucsc.edu/cgi-bin/hgLiftOver
可以看到4个外显子坐标成功转换完成，点击View conversions就可以得到转换完成的hg38的外显子坐标，外显子坐标为：
chr1:228106685-228106918
chr1:228107668-228107869
chr1:228108235-228108318
chr1:228109261-228109338
将相应位置的DNA序列与LncRNA序列比对
可以看到LNC_000332一共匹配了4段，染色体坐标分别为chr1:228106685-228106919，chr1:228107668-228107870，chr1:228108235-228108332，chr1:228109259-228109938
而外显子的染色体坐标分别为chr1:228106685-228106918，chr1:228107668-228107869，chr1:228108235-228108318，chr1:228109261-228109338
这里可以看到LNC_000332的序列基本都在外显子里面，所以不适合选择这个LncRNA来做PCR验证