southern blotting
synthesizes DNA
northern blotting
synthesizes RNA
western blotting
synthesizes protiens
1st step PCR
Primers target a specific region
2nd step PCR
Denature DNA (~95°C)
3rd step PCR
Anneal primers (~55°-65°C)
1st step cDNA synthesis
RNA purification
2nd step cDNA synthesis
RNA incubated with reverse transcriptase
3rd step of cDNA synthesis
oligo-dT primer anneals polyA tail of mRNA so reverse transcriptase synthesizes single strands DNA
4th step of cDNA synthesis
mRNA is removed and DNA polymerse copies the cDNA strand
5th step of cDNA synthesis
nick hairpin
4th step PCR
Elongate new DNA strands, using Taq or other DNA polymerase(~72°C)
5th step PCR
Repeat denaturation, annealing, and elongation for 20-40 cycles.
miRNA and siRNA similarities
21-22 nucleotides
undergo post-transcriptional processing.
involve the formation
of double-stranded RNA
participate in RNA interference (RNAi), targeted, RNA-
mediated
repression of gene expression
siRNAs
...
miRNA
Many are produced by RNA pol II
regulate other genes
may have a mismatch with target transcripts
are implicated in developmental processes
may block translation and/or trigger RNA degradation