limit of detection with RT-qPCR (SYBR or TaqMan) - 未名空间MITBBS历史存档

limit of detection with RT-qPCR (SYBR or TaqMan)# Biology - 生物学

y*u2013-09-06 07:09

1 楼

发20个包子吧。先发个上次给祝福的。
485 timeline:
RD: 8/13;
ND: 9/8;
FD: 10/4;
Approved on 11/8.
上次求了祝福的，等会我把剩的包子发了啊。
“Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
ADJUST STATUS
Your Case Status: Card/ Document Production
On November 8, 2010, we ordered production of your new card. Please allow 30
days for your card to be mailed to you. If we need something from you we
will contact you. If you move before you receive the card, call customer
service at 1-800-375-5283. ”

x*a2013-09-06 07:09

2 楼

The children were lined up in the cafeteria of a Catholic elementary school
for lunch. At the head of the table was a large pile of apples. The nun made
a note, and posted on the apple tray:
'Take only ONE. God is watching.'
Moving further along the lunch line, at the other end of the table was a
large pile of chocolate chip cookies.
A child had written a note, 'Take all you want. God is watching the apples.

l*82013-09-06 07:09

3 楼

第一段视频：http://v.youku.com/v_show/id_XODAzNjkzNzk2.html
第二段视频：http://v.youku.com/v_show/id_XODA0NjgwODIw.html?f=22944973&from=y1.1-2.10027-0.1-1
长期被虐待的女宝需要高手帮助，跪求大家帮查ip 地址，

a*a2013-09-06 07:09

4 楼

Here is a technology question. I know it should be possible to improve the
result and would like to hear some more suggestions.
Basically we are trying to push the limit of detection to the level of 10e4
or even 10e3, for viral RNA isolated from animal blood samples, for
pharmacokinetic study.
Our samples contain mostly carrier RNA with viral RNA of interest.
And with SBYR or TaqMan, we can stably detect 200-300 copies per well (of 96
-well setting), that translates into 10e5 copies/ml with our settings.
In theory, it seems it should be able to go as low as 50 copies per well. Or
even lower, if the samples are pure gene of interest, no other matrix
system (such as carrier RNA).
So my specific questions here are, for people who are doing about the same
research:
1. How you prepare your samples?
2. what's the limit of detection you get to with SYBR or Taqman?
BioRad claims that ddPCR can go low to 10 copies per well. It might have a
much even better chance to get precise and accurate result with samples we
have (lots of carrier RNA). But I am just curious, if we don't want to use
ddPCR, but just regular bioRad qPCR machine, how low the concentration can
go to in terms of limit of detection? And how people prepare their sample to
reach this goal?
Any input is appreciated. many thanks.

u*g2013-09-06 07:09

5 楼

gxgx

a*h2013-09-06 07:09

6 楼

not bad

l*82013-09-06 07:09

7 楼

Youtube link http://youtu.be/thH4keTuqYc please support !

C*e2013-09-06 07:09

8 楼

limit应该是PCR Efficiency of certain primer pair
我以前做absolute quantification的时候，standard curve的dynamic range是10^6到
10^2
standard curve出来很好，从Ct值上判断再多一个1/10 dilution也是可以的
PCR Efficiency能够到1.8、1.9的话，测50个拷贝应该不是什么问题
但是现在大多数做qPCR的实验根本就不去测每对primer pair的PCR Efficiency

c*e2013-09-06 07:09

9 楼

GXGX!

h*k2013-09-06 07:09

10 楼

哈哈

s*92013-09-06 07:09

11 楼

When you deal with less than 100 copies, you need to consider RT-PCR as a
series of random processes (e.g., Poisson): 1) template destruction, which
is significant for RNA templates, 2) distribution of successful cDNA
synthesis, and 3) distribution of successful cDNA amplification in each
cycle. Many stochastic variations can affect a final output.

z*a2013-09-06 07:09

12 楼

gx

a*a2013-09-06 07:09

13 楼

Thanks for the input.
Chamgrape, we might be using different system, the way how we calculate and
express the PCR efficiency is by %. Mine's usually between 90-100% (good
range is 80% - 110%). What kind of samples you are using for "no problem
with 50 copies/well?". Are you using pure sample or you have to harvest the
samples with a matrix system (such as carrier RNA etc).
synbio79, i was wondering how much it can help to optimize the RT experiment
before doing qPCR. Have you done so?

L*y2013-09-06 07:09

14 楼

GXGX

C*e2013-09-06 07:09

15 楼

我不知道这个80%-110%是怎么计算的
Bio-Rad有些好像是这样显示PCR Efficiency的
我说的就是自己做曲线，用质粒，因为浓度、大小（分子量）已知，然后推导
如果你说的80%-110%跟Bio-Rad家的一样，那么是还可以啦。
我说的case是cDNA，我没有做过carrier RNA

and
the
experiment

【在 a******a 的大作中提到】

: Thanks for the input.
: Chamgrape, we might be using different system, the way how we calculate and
: express the PCR efficiency is by %. Mine's usually between 90-100% (good
: range is 80% - 110%). What kind of samples you are using for "no problem
: with 50 copies/well?". Are you using pure sample or you have to harvest the
: samples with a matrix system (such as carrier RNA etc).
: synbio79, i was wondering how much it can help to optimize the RT experiment
: before doing qPCR. Have you done so?

s*o2013-09-06 07:09

16 楼

恭喜恭喜！

d*o2013-09-06 07:09

17 楼

恭喜恭喜！

s*82013-09-06 07:09

18 楼

cool

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

R*n2013-09-06 07:09

19 楼

GXGX

m*s2013-09-06 07:09

20 楼

congratulations

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

s*t2013-09-06 07:09

21 楼

yeah, a typical time frame, 3 months.
Congratulations.

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

P*e2013-09-06 07:09

22 楼

恭喜恭喜！

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

s*s2013-09-06 07:09

23 楼

恭喜恭喜月如妹妹。
FD之后5周批准，看来我还得等上两周左右。

TO
allow 30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

C*12013-09-06 07:09

24 楼

GXGX

S*92013-09-06 07:09

25 楼

GXGX
包子
我是22打的指纹，上个月，看来这个月有希望了

c*n2013-09-06 07:09

26 楼

cong!!!

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

z*32013-09-06 07:09

27 楼

恭喜啊，有包子吃没？
^_^

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

b*M2013-09-06 07:09

28 楼

恭喜恭喜！恭喜恭喜！恭喜恭喜！

g*e2013-09-06 07:09

29 楼

gxgx

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

l*t2013-09-06 07:09

30 楼

congrats

s*r2013-09-06 07:09

31 楼

GXGX, Paibaozi

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

A*d2013-09-06 07:09

32 楼

恭喜恭喜月如妹妹, Dian 喜 qi.

u*92013-09-06 07:09

33 楼

gxgx

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

p*i2013-09-06 07:09

34 楼

gxgx

H*e2013-09-06 07:09

35 楼

GXGX!

b*a2013-09-06 07:09

36 楼

gxgx!

M*n2013-09-06 07:09

37 楼

gxgx！

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

d*12013-09-06 07:09

38 楼

Congrats!!

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

d*12013-09-06 07:09

39 楼

Congrats!!

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

G*n2013-09-06 07:09

40 楼

GXGX!

w*d2013-09-06 07:09

41 楼

GXGX

j*n2013-09-06 07:09

42 楼

cong

Y*12013-09-06 07:09

43 楼

cong......baozi please

x*h2013-09-06 07:09

44 楼

GXGX

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

l*82013-09-06 07:09

45 楼

Cong!

z*o2013-09-06 07:09

46 楼

gxgx

t*n2013-09-06 07:09

47 楼

GXGX!!

d*22013-09-06 07:09

48 楼

cong

485 timeline:
RD: 8/13;
ND: 9/8;
FD: 10/4;
Approved on 11/8.
上次求了祝福的，等会我把剩的包子发了啊。
“Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
ADJUST STATUS
Your Case Status: Card/ Document Production
On November 8, 2010, we ordered production of your new card. Please allow 30
days for your card to be mailed to you. If we need something from you we
will contact you. If you move before you receive the card, call customer
service at 1-800-375-5283. ”

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

m*d2013-09-06 07:09

49 楼

gxgx

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

b*y2013-09-06 07:09

50 楼

congratulations!!

y*o2013-09-06 07:09

51 楼

恭喜！

c*k2013-09-06 07:09

52 楼

gx

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

L*r2013-09-06 07:09

53 楼

恭喜

g*92013-09-06 07:09

54 楼

GXGX!!!!

v*92013-09-06 07:09

55 楼

Much faster than mine. GXGX

C*r2013-09-06 07:09

56 楼

gxgx

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

s*p2013-09-06 07:09

57 楼

gxgx

s*u2013-09-06 07:09

58 楼

[email protected]!!!

wh2013-09-06 07:09

59 楼

这个id很眼熟，好像是校版的？祝贺。

TO
30

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

f*t2013-09-06 07:09

60 楼

gxgx

c*b2013-09-06 07:09

61 楼

re

m*n2013-09-06 07:09

62 楼

GX GX

y*u2013-09-06 07:09

63 楼

是的。你的id偶也很熟的。
军训过的。

【在 wh 的大作中提到】

: 这个id很眼熟，好像是校版的？祝贺。
:
: TO
: 30

c*t2013-09-06 07:09

64 楼

Con

v*k2013-09-06 07:09

65 楼

gxgx
沾喜气

TO
30

【在 y***u 的大作中提到】

: 是的。你的id偶也很熟的。
: 军训过的。

b*y2013-09-06 07:09

66 楼

恭喜恭喜！

y*h2013-09-06 07:09

67 楼

gx, 沾点喜气

a*e2013-09-06 07:09

68 楼

cong!

d*e2013-09-06 07:09

69 楼

GXGX!

r*g2013-09-06 07:09

70 楼

re

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

L*r2013-09-06 07:09

71 楼

恭喜

h*52013-09-06 07:09

72 楼

GXGX!

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

c*62013-09-06 07:09

73 楼

GXGX!

b*92013-09-06 07:09

74 楼

gx

a*52013-09-06 07:09

75 楼

恭喜恭喜，挺顺利的。

G*02013-09-06 07:09

76 楼

GXGX

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

e*22013-09-06 07:09

77 楼

恭喜噢！

N*82013-09-06 07:09

78 楼

Gong Gong

c*b2013-09-06 07:09

79 楼

gxgx

k*n2013-09-06 07:09

80 楼

gxgx

z*b2013-09-06 07:09

81 楼

cong

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

E*02013-09-06 07:09

82 楼

gxgx

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

y*a2013-09-06 07:09

83 楼

GXGX

y*n2013-09-06 07:09

84 楼

Cong~. How long? 10 year?

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

k*i2013-09-06 07:09

85 楼

cong

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

J*n2013-09-06 07:09

86 楼

congratulations.

J*n2013-09-06 07:09

87 楼

congratulations.

f*r2013-09-06 07:09

88 楼

GXGX

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production

L*62013-09-06 07:09

89 楼

GX, GX

TO

【在 y***u 的大作中提到】

: 发20个包子吧。先发个上次给祝福的。
: 485 timeline:
: RD: 8/13;
: ND: 9/8;
: FD: 10/4;
: Approved on 11/8.
: 上次求了祝福的，等会我把剩的包子发了啊。
: “Application Type: I485 , APPLICATION TO REGISTER PERMANENT RESIDENCE OR TO
: ADJUST STATUS
: Your Case Status: Card/ Document Production