Hosuk:LabNotes/2013-12-30

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RT primer annealing specific position in mRNA[edit]

Primer Design[edit]

  • Target gene : RAB7A
  • RAB7A mRNA target sequence: CAGAACTTGGACCTTCTCGCTTCTGTCCTCCGTTTAGTCTCCTC
    • This is base 125-177
  • Choose primers that are 10bp long while minimizing GC content and ~200bp, ~300bp, ~500bp, and ~1000bp from TSS


  • mRNA target of RAB7A 220b distance from TSS : CGCGTTTGAA
  • mRNA target of RAB7A 300b distance from TSS : ACTCATGAAC
  • mRNA target of RAB7A 510b distance from TSS : CAACACATTC
  • mRNA target of RAB7A 1000b distance from TSS: TTACACCCCA


  • RT_RAB7A_220b: [/5Phos/TCTCGGGAACGCTGAAGA] + TTCAAACGCG (--> D220)
  • RT_RAB7A_300b: [/5Phos/TCTCGGGAACGCTGAAGA] + GTTCATGAGT (--> D300)
  • RT_RAB7A_510b: [/5Phos/TCTCGGGAACGCTGAAGA] + GAATGTGTTG (--> D510)
  • RT_RAB7A_1000b: [/5Phos/TCTCGGGAACGCTGAAGA] + TGGGGTGTAA(--> D1000)


Rolony Fab.[edit]

  • Use 96 well plate (12/28~12/30)
    • Control : Random Hexamer

File:PGP1F 96well RTpositionTest Cells 2.png


Result[edit]

  • Rolonies from 1000b primer were too many, they are much more than control (random hexamer).
  • There is a trend, 220b and 300b primer sample don't have many rolonies which are almost similar number that we've seen so far with RAB7A detection.
  • However 500b sample showed more rolonies, and 1000b sample has too many and too much strong signal.
  • I'm not sure the signal from 1000b sample is real signal or something other things...because it's too much.
  • But since I've made 4 wells per each sample, and all 4 wells have the sample result per each primer, so I think these images shows some meaningful results.


  • 1st Rolony Counts

File:PGP1F 96well RTpositionTest 1stRolonyCount.png


  • PISA parameters
    • Gaussian Std : 3
    • Gaussian Upper : -2e-4
    • area upper: 50
    • area lower: 4
    • axratio lower: 0.6
    • circ upper: 1.6
    • circ lower: 0.8
    • perim conn: 8
    • bkgmult lower: 4


Result : RAB7A Target on 1st Rolony[edit]

  • ATTO550-RAB7A on 1st Rolony probes : NO signal at all! WHY?
    • I annealed Cy3 1st Rolony probes : showed the same signals to the previous Cy5 1st Rolony probes… so there are still 1st Rolonies…BUT then why there are no 1st Rolonies with RAB7A targets?