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Home > ÀüÁ¦Ç°º¸±â > Real Time PCR > Cell direct qPCR > RNA ÃßÃâ¾øÀÌ cell direct qPCR
1.5½Ã°£ÀÌ¸é ¹è¾ç¼¼Æ÷¿¡¼­ Real Time PCR °á°ú È®ÀÎ

RNA ÃßÃâ¾øÀÌ cell direct qPCR

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
3735S
CellAmp¢â Direct TB Green¢ç RT-qPCR Kit
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
252,000¿ø 
315,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3735A_e.v2111Da.pdf, 3735S_e.v2111Da.pdf
Takara
3735A
CellAmp¢â Direct TB Green¢ç RT-qPCR Kit
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
948,800¿ø 
1,186,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
Takara
3736A
CellAmp¢â Direct Probe RT-qPCR Kit
°ü·ÃÇмú ±¸¸ÅÇϱâ
1 Kit
886,400¿ø 
1,108,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3736A_e.v2111Da.pdf, 3736S_e.v2111Da.pdf
Takara
3737S
CellAmp¢â Direct Lysis and RT set
°ü·ÃÇмú ±¸¸ÅÇϱâ
1 Set (20 ȸ)
151,200¿ø 
189,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3735S_e.v1809Da.pdf, 3736S_e.v1904Da.pdf
Takara
3737A
CellAmp¢â Direct Lysis and RT set
°ü·ÃÇмú ±¸¸ÅÇϱâ
1 Set (100 ȸ)
607,200¿ø 
759,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
Takara
3738A
CellAmp¢â Direct Lysis set
°ü·ÃÇмú ±¸¸ÅÇϱâ
1 Kit (Lysis 100ȸ)
281,600¿ø 
352,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3735S_e.v1809Da.pdf, 3736S_e.v1904Da.pdf
Takara
3739A
CellAmp¢â Direct RT Enzyme set
°ü·ÃÇмú ±¸¸ÅÇϱâ
1 Kit (RT 100ȸ)
486,400¿ø 
608,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3735S_e.v1809Da.pdf, 3736S_e.v1904Da.pdf

Á¦Ç° ¼³¸í
º» Á¦Ç°Àº 96 well ȤÀº ´Ù¾çÇÑ ÇüÅÂÀÇ plate¿¡¼­ ¹è¾çµÈ µ¿¹°¼¼Æ÷ (adherent cells, non-adherent cells, primary cells, stem cells, iPS cells µî)·ÎºÎÅÍ RNA ÃßÃâ °úÁ¤ ¾øÀÌ °£´ÜÇÑ ¹æ¹ýÀ¸·Î 2-step Real Time RT-PCRÀ» ¼öÇà ÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°À» »ç¿ëÇÏ¸é ¹è¾ç¼¼Æ÷¿¡¼­ template RNA Á¶Á¦, ¿ªÀü»ç¹ÝÀÀ, Real Time PCR±îÁö ¾à 1.5½Ã°£ÀÌ ¼Ò¿äµÇ¸ç, genomic DNA¸¦ È¿°úÀûÀ¸·Î Á¦°ÅÇÒ ¼ö Àֱ⠶§¹®¿¡ genomic DNAÀÇ È¥ÀÔÀÌ ºÐ¼®¿¡ ¿µÇâÀ» ¹ÌÄ¡´Â °æ¿ì (Exon Á¢ÇÕÀÇ primer µðÀÚÀÎÀÌ ºÒ°¡´ÉÇÑ °æ¿ì, Àú¹ßÇö À¯ÀüÀÚ¸¦ Çؼ®ÇÏ´Â °æ¿ì µî)¿¡µµ À¯¿ëÇÏ´Ù.
Intercalater (TB Green¢ç) °ËÃâ¹ý ¶Ç´Â Dual Labeled-probe °ËÃâ¹ýÀ» À§ÇÑ 2 step Real Time RT-PCR ½Ã¾àÀÌ ÇÔ²² ±¸¼ºµÇ¾î ÀÖ¾î, Æí¸®ÇÏ°Ô À¯ÀüÀÚ ¹ßÇö Çؼ®À» ÁøÇàÇÒ ¼ö ÀÖ´Ù.
* #3737S/A´Â #3735S/A, #3736S/AÀÇ ±¸¼ºÇ°ÀÔ´Ï´Ù.
ÀÚ¼¼ÇÑ Á¦Ç°»ç¿ëÀº #3735 ¶Ç´Â #3736ÀÇ ¸Å´º¾ó VI. Protocol, VI-1. ~ VI-3. (Cell lysis ~ Reverse Transcription Reaction)À» Âü°í ¹Ù¶ø´Ï´Ù.
* #3735S/A, #3736S/AÀÇ Real Time PCR ½Ã¾àÀº ¾Æ·¡ÀÇ Á¦Ç°À¸·Î ±¸¸ÅÇÏ½Ç ¼ö ÀÖ½À´Ï´Ù.
- 3735A/BÀÇ °æ¿ì: TB Green¢ç Fast qPCR Mix (Code RR430A/B)
- 3736A/BÀÇ °æ¿ì: Probe qPCR Mix (Code RR391A/B)
Á¦Ç° Ư¡
1. ¾à 1.5½Ã°£¿¡ °íÈ¿À²·Î À¯ÀüÀÚ ¹ßÇö ºÐ¼® °¡´É
º» Á¦Ç°À» »ç¿ëÇϸé Real Time PCR ÁÖÇüÀ» RNAÀÇ Á¤Á¦ °úÁ¤ ¾øÀÌ ¼¼Æ÷·ÎºÎÅÍ Á÷Á¢ Áغñ°¡ °¡´ÉÇÏ´Ù. ¶ÇÇÑ, ±âÁ¸ÀÇ Á¦Ç° (CellAmp¢â Direct RNA Prep Kit for RT-PCR (Real Time) Code 3732)¿Í ´Þ¸®, ¿ªÀü»ç °úÁ¤ Àü DNase inactivationÀ» À§ÇÑ ¿­Ã³¸® °úÁ¤ÀÌ ÇÊ¿ä ¾ø´Ù. ¶ÇÇÑ ½ÇÇè ÀýÂ÷°¡ °£´ÜÇÏ¿©, °íÈ¿À²(high-throughput)ºÐ¼®¿¡ ÀûÇÕÇÑ Á¦Ç°ÀÌ´Ù.
2. Áٱ⼼Æ÷(¿¹: iPS cells)·Î ºÐÈ­µÈ ¼¼Æ÷ÀÇ ºÐ¼®½Ã ÀûÇÕ
º» Á¦Ç°Àº Çâ»óµÈ Lysis Buffer II¸¦ »ç¿ëÇÏ¿© ´Ù¾çÇÑ ÇüÅÂÀÇ ¼¼Æ÷ÁÖ(adherent cells, non-adherent cells, primary cells, stem cells, iPS cells, etc)¿¡ »ç¿ë °¡´ÉÇÏ´Ù.
3. ¼¼Æ÷ ¿ëÇع°(Cell lysates) Àå±â°£ º¸°ü °¡´É
º» Á¦Ç°À¸·Î Á¦Á¶ÇÑ ¼¼Æ÷ ¿ëÇع°Àº -20¡É¿¡¼­ ÃÖ´ë 6°³¿ù º¸°ü °¡´ÉÇÏ´Ù.
4. ³ôÀº Á¤È®µµ¿Í PCR ¾ïÁ¦¹°Áú¿¡ ´ëÇÑ ÀúÇ×¼º
º» Á¦Ç°¿¡ »ç¿ëµÈ TB Green¢ç Fast qPCR Mix (Code RR430)¿Í Probe qPCR Mix (Code RR391)Àº PCR ¾ïÁ¦¹°Áú¿¡ °­·ÂÇÑ ÀúÇ×¼ºÀ» °¡Áö°í ÀÖÀ¸¸ç, GC-rich template¿¡¼­µµ ³ôÀº Á¤È®µµ¸¦ ³ªÅ¸³½´Ù.
Work flow

³»¿ë

¡¡

±¸¼ºÇ°¸í

3735A / 3736A

3737A

3738A

3739A

3735S / 3736S

3737S

1.

CellAmp Washing Buffer*1

2.5 §¢ ¡¿ 5

2.5 §¢ ¡¿ 5

2.5 §¢ ¡¿ 5

-

2.5 §¢

2.5 §¢

2.

CellAmp Lysis Buffer II*1

1 §¢ ¡¿ 5

1 §¢ ¡¿ 5

1 §¢ ¡¿ 5

-

1 §¢

1 §¢

3.

DNase I for Direct RNA Prep*1

200 §¡

200 §¡

200 §¡

-

40 §¡

40 §¡

4.

Stop Solution*1

250 §¡

250 §¡

250 §¡

-

50 §¡

50 §¡

5.

PrimeScript RT Enzyme Mix*2

200 §¡

200 §¡

-

200 §¡

40 §¡

40 §¡

6.

5 ¡¿ CellAmp Buffer II*2

400 §¡

400 §¡

-

400 §¡

80 §¡

80 §¡

7.

RT Primer Mix*2,3

100 §¡

100 §¡

-

100 §¡

20 §¡

20 §¡

8.

RNase Free H2O*2

1 §¢ ¡¿ 2

1 §¢ ¡¿ 2

-

1 §¢ ¡¿ 2

1 §¢

1 §¢

9.

TB Green¢ç Fast qPCR Mix (2¡¿) (3735A/S)*4
¶Ç´Â Probe qPCR Mix (2¡¿) (3735A/S)*4

625 §¡ ¡¿ 10

-

-

-

625 §¡ ¡¿ 2

-

10.

ROX Reference Dye (50¡¿)*5

250 §¡

-

-

-

50 §¡

-

11.

ROX Reference Dye II (50¡¿)*5

250 §¡

-

-

-

50 §¡

-


*1

Cell Lysis ¿ë ½Ã¾à

3735A / 3736A / 3737A      100 ȸ

3735S / 3736S / 3737S      20 ȸ

*2

RT (¿ªÀü»ç) ½Ã¾à

3735A / 3736A / 3737A      100 ȸ

3735S / 3736S / 3737S      20 ȸ

*3

Oligo dT Primer, Random 6 mers¸¦ Æ÷ÇÔÇÑ´Ù.

*4

qPCR ½Ã¾à

3735A / 3736A      500 ȸ

3735S / 3736S      100 ȸ

 *5   Life TechnologiesÀÇ Real Time PCR ÀåÄ¡ µî well °£ Çü±¤ ½ÅÈ£¸¦ º¸Á¤ÇÏ´Â ÀåÄ¡·Î Çؼ®ÇÏ´Â °æ¿ì¿¡ »ç¿ëÇϸç, ÀÚ¼¼ÇÑ ³»¿ëÀº Á¦Ç°¸Å´º¾óÀ» Âü°í.
iPS ¼¼Æ÷ À¯·¡ ½É±Ù¼¼Æ÷¸¦ »ç¿ëÇÑ ½ÇÇè ¿¹
CellAmp¢â Direct TB Green¢ç RT-PCR Kit (Code 3735)ÀÇ ½ÇÇè ¿¹
<½ÇÇè¹æ¹ý> iPS cellsÀ» Cardiomyocyte·Î ºÐÈ­ À¯µµÇϸç 5ÀÏ, 18ÀÏ, 42ÀÏ ÈÄ (°¢°¢ day 5, day 18, day 42·Î Ç¥±â), 1x104 cellsÀ» harvestÇÏ¿© º» Á¦Ç°ÀÇ ÇÁ·ÎÅäÄÝ¿¡ µû¶ó cell lysate 50 §¡¸¦ Á¶Á¦ÇÏ¿´´Ù.
Á¦½ÃµÈ ÇÁ·ÎÅäÄÝ¿¡ µû¶ó ¿ªÀü»ç ¹ÝÀÀ°ú Real-Time PCRÇÏ¿© À¯ÀüÀÚ ¹ßÇö ºÐ¼®À» ÁøÇàÇÏ°í,
Á¤Á¦µÈ total RNA (1x104 cells¿¡¼­ ÃßÃâ) ¸¦ control·Î »ç¿ëÇÏ¿© µ¿ÀÏÇÑ ¹æ½ÄÀ¸·Î À¯ÀüÀÚ ¹ßÇö ºÐ¼®À» ÁøÇàÇÏ¿´´Ù.


<°á°ú> Day 5ÀÇ À¯ÀüÀÚ ¹ßÇö·®À» 1·Î ÇÏ¿© Day 18°ú Day 42ÀÇ À¯ÀüÀÚ ¹ßÇö·®À» ºñ±³ÇÑ °á°ú, º» Á¦Ç°¿¡¼­ Á¶Á¦ÇÑ cell lysate°¡ Á¤Á¦µÈ RNA¸¦ »ç¿ëÇÑ °æ¿ì¿Í µ¿µîÇÑ À¯ÀüÀÚ ¹ßÇö profileÀ» ³ªÅ¸³¿À» È®ÀÎÇÏ¿´´Ù.

CellAmp¢â Direct Probe RT-qPCR Kit (Code 3736)ÀÇ ½ÇÇè ¿¹
<½ÇÇè¹æ¹ý> ´Ù¾çÇÑ ¼¼Æ÷ (HeLa, Jurkat, 239, normal human dermal fibrolast (NHDF))¿Í iPS cell·ÎºÎÅÍ ºÐÈ­µÈ cardiomyocyte (CM)¸¦ °¢°¢ 1x104 cells·Î harvestÇÏ¿© º» Á¦Ç°ÀÇ ÇÁ·ÎÅäÄÝ¿¡ µû¶ó cell lysate 50 §¡¸¦ Á¶Á¦ÇÏ¿´´Ù.
Á¦½ÃµÈ ÇÁ·ÎÅäÄÝ¿¡ µû¶ó ¿ªÀü»ç ¹ÝÀÀ°ú Real-Time PCRÇÏ¿© À¯ÀüÀÚ ¹ßÇö ºÐ¼®À» ÁøÇàÇÏ°í,
Á¤Á¦µÈ total RNA (1x104 cells¿¡¼­ ÃßÃâ) ¸¦ control·Î »ç¿ëÇÏ¿© µ¿ÀÏÇÑ ¹æ½ÄÀ¸·Î À¯ÀüÀÚ ¹ßÇö ºÐ¼®À» ÁøÇàÇÏ¿´´Ù.


<°á°ú> º» Á¦Ç°¿¡¼­ Á¶Á¦ÇÑ cell lysate°¡ Á¤Á¦µÈ RNA¸¦ »ç¿ëÇÑ °æ¿ì¿Í µ¿µîÇÑ À¯ÀüÀÚ ¹ßÇö profileÀ» ³ªÅ¸³¿À» È®ÀÎÇÏ¿´´Ù.
º¸Á¸ -20¡É
CellAmp Washing Buffer ¹× CellAmp Lysis Buffer II
À¶ÇØ ÈÄ 4¡É¿¡ º¸°üÇϽðí, ¿À¿°¿¡ ÁÖÀÇ ÇϽʽÿÀ.

3735S / A : TB Green¢ç Fast qPCR Mix(2x)
4¡É º¸°ü½Ã 6°³¿ù ¾ÈÁ¤Çϸç, ¹Ýµå½Ã Â÷±¤ÇÏ¿© º¸°ü ÇϽʽÿÀ.
- Àå±â º¸Á¸ÀÇ °æ¿ì´Â -20 ¡É¿¡¼­ º¸°üÇϽʽÿÀ. Çѹø Çص¿ÇÑ TB Green¢ç Fast qPCR Mix (2 x)´Â 4¡É º¸°üÇÏ°í 6°³¿ù ¾È¿¡ »ç¿ëÇÒ °ÍÀ» ±ÇÀåÇÕ´Ï´Ù.
- »ç¿ëÀü ¿ÏÀüÈ÷ ³ìÀÎ ÈÄ ±ÕÀÏÇÏ°Ô È¥ÇÕÇÏ¿© »ç¿ëÇϽñ⠹ٶø´Ï´Ù.

3736S / A : Probe qPCR Mix(2x)
4¡É º¸°ü½Ã 6°³¿ù ¾ÈÁ¤Çϸç, ¹Ýµå½Ã Â÷±¤ÇÏ¿© º¸°ü ÇϽʽÿÀ.
- Àå±â º¸Á¸ÀÇ °æ¿ì´Â -20 ¡É¿¡¼­ º¸°üÇϽʽÿÀ. Çѹø Çص¿ÇÑ Probe qPCR Mix (2 x)´Â 4¡É º¸°üÇÏ°í 6°³¿ù ¾È¿¡ »ç¿ëÇÒ °ÍÀ» ±ÇÀåÇÕ´Ï´Ù.
- »ç¿ëÀü ¿ÏÀüÈ÷ ³ìÀÎ ÈÄ ±ÕÀÏÇÏ°Ô È¥ÇÕÇÏ¿© »ç¿ëÇϽñ⠹ٶø´Ï´Ù.

Keyword : cell to ct,crude qPCR,¹è¾ç¼¼Æ÷ qPCR,real time PCR,½Ç½Ã°£PCR

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