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===RT primer annealing specific position in mRNA=== ====Primer Design==== *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.==== *Use 96 well plate (12/28~12/30) **Control : Random Hexamer [[File:PGP1F_96well_RTpositionTest_Cells_2.png|450px]] ====Result==== *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. *Raw images : [[Media:MIP_RTPrimer_RAB7A_C3_Hexamer-1_Step1_Pos1.tif|Control]], [[Media:MIP_RTPrimer_RAB7A_D3_D220-1_Step1_Pos1.tif|D220]], [[Media:MIP_RTPrimer_RAB7A_D7_D300-1_Step1_Pos1.tif|D300]], [[Media:MIP_RTPrimer_RAB7A_E3_D510-1_Step1_Pos1.tif|D510]], [[Media:MIP_RTPrimer_RAB7A_E7_D1000-1_Step1_Pos1.tif|D1000]] *[[File:MIP_RTPrimer_1stRolony_Compare.png|800px]] *1st Rolony Counts [[File:PGP1F_96well_RTpositionTest_1stRolonyCount.png|800px]] *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==== *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?
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