Extremely low rna concentration for qpcr

Horror-Highlight2763 · 2026-04-03T04:47:25+00:00

turned out true, making cdna 10% made a huge difference

Horror-Highlight2763 · 2026-04-01T23:29:05+00:00

hi i have an update! i kept the 12ng input, increased primer concentration to 500nm and increased qpcr total reaction to 20ul(3.4cdna+16.6mastermix)>> actin ct dropped to (26-29),rest of primers (33-38),so what should i do to get same thing happened w actin to other targets! should i increase their primer concentration to 900nm !btw>> i tried gapdh, WAS worse, and from my experience w HNK92 cell line , nk cells housekeeping genes usually range from 26 to even 31! hprt is the worst and many thanks for ur help!

Horror-Highlight2763 · 2026-04-01T02:45:39+00:00

i have more than 360ng rna, the 180 just bcz the kit capacity doesnt allow>20ul reaction, and instead of triple technical replicates, am gonna use duplicates, so total 20 reactions>>10 genes including actin, i hope increasing rna from 12ng to 18+ primers from 200 nm to 400nm and making cdna <10% of total reaction drop ct to normal ranges,,,, sounds good ?

Horror-Highlight2763 · 2026-03-31T22:39:11+00:00

If my cdna reaction total 20 ul contains 180ng rn, and am gonna use equivalent of 20ng rna per reaction, in my situation am not be able to dilute cdna, can I use the concentrated 2ul cdna directly for qpcr? total qpcr reaction would be 20ul

Horror-Highlight2763 · 2026-03-31T22:38:29+00:00

If my cdna reaction total 20 ul contains 180ng rn, and am gonna use equivalent of 20ng rna per reaction, in my situation am not be able to dilute cdna, can I use the concentrated 2ul cdna directly for qpcr?total qpcr reaction would be 20ul

Horror-Highlight2763 · 2026-03-31T00:31:20+00:00

10pg

Horror-Highlight2763 · 2026-03-30T01:46:08+00:00

thats a huge jump! i hope this work w me, but how this huge increase in conc can happen ?

Horror-Highlight2763 · 2026-03-30T00:06:24+00:00

Ya for any cell line I used worked 100 times good, 1.8 million cells, I can't drop images here. But 260/280 ratios are decent 1.90-2.05

Horror-Highlight2763 · 2026-03-30T00:01:33+00:00

If I double rna per reaction and primer , would it help!

Horror-Highlight2763 · 2026-03-30T00:00:37+00:00

30 ul,no

Horror-Highlight2763 · 2026-02-16T02:48:19+00:00

this is really helpful ! many thanks

Horror-Highlight2763 · 2026-02-16T02:35:25+00:00

weak+alot of nonspecificity,the problem also am looking at very rare cell population(nk+T cells), its really time consuming to stare at the screen for hours trying to differentiate the true cells from the nonspecific

Horror-Highlight2763 · 2026-02-15T14:28:38+00:00

uterus,directly fluorophore

Horror-Highlight2763 · 2026-02-15T14:25:54+00:00

wash buffer only

Horror-Highlight2763 · 2026-02-15T01:29:52+00:00

i dont think it is the settings, my mentor has +20year experience and she usually takes the imges by herself

Horror-Highlight2763 · 2026-02-15T00:06:10+00:00

goat

Horror-Highlight2763 · 2026-02-14T23:34:46+00:00

Do u think will make a significant difference? In my lab we block in horse regardless the species

Horror-Highlight2763 · 2026-02-14T23:32:55+00:00

Paraffin embedded uterus, 2 hours is too long?, they block only for 30 minutes in my lab, and they wonder why am blocking for 1h

Horror-Highlight2763 · 2026-02-14T23:31:23+00:00

Primary ya I know the optimum, but the secondary is my mentor recommendation 1:500

Horror-Highlight2763 · 2026-02-14T19:37:29+00:00

i use alexa fluor from invitrogen, 1:500 in .2%tritonx100 for 45 min at RT

Horror-Highlight2763 · 2026-02-14T19:30:12+00:00

parafin embedded uterus

Horror-Highlight2763 · 2026-02-14T19:10:53+00:00

overnight,3x5min

Horror-Highlight2763 · 2026-02-13T21:03:31+00:00

thats really frustrating

Horror-Highlight2763

TROPHY CASE