서비스 문의 상담

바이오코아(주) TEL. 02-853-4605 / syyu@bio-core.com

다카라코리아 TEL. 02-2081-2510 / support@takara.co.kr

•One-stop shop: 상담부터 array 실험, 데이터 분석까지

•상동성이 매우 높은 microRNA family도 특이적으로 구별

•고감도: 30 ng 수준의 total RNA로부터 분석 가능

(추천 샘플량 : Total RNA 250 ng - 1,000 ng)

•FFPE 유래의 microRNA microarray 분석

하나! Array 서비스를 의뢰하시면 validation을 위한 miRNA qPCR 제품을 30% 할인가로 드립니다.

둘! 재의뢰시 사용할 수 있는 서비스가격 할인쿠폰을 드려요.

Special Offer !!!

LNA™를 이용한 microarray결과는 뒷면을 확인하세요.

* 본 microRNA microarray 서비스는 다카라코리아의 협력회사인 바이오코아(주)를 통하여 진행됩니다.

* 서비스 소요시간은 샘플조건 등 상황에 따라 달라질 수 있습니다.

Consultation &

experimental design

Report &

final consultation

RNA sample

submission

Data analysis &

normalization

RNA sample

quality control (QC)

Labeling, hybridization,

scanning

Casino Chip???Potato Chip???

Micro Chip???

•miRBase기반의 human, mouse, rat microRNA검출

•LNA™-enhanced probe에 의한

높은 특이성과 민감도

miRCURY LNA™ microRNA Array, 7th gen

파격 할인!

샘플당 60만원!

기간: 8월3일~11월30일

MicroRNAMicroarray 분석서비스

더 파격적이고 더 재미있는 RNA Chip!

[MicroRNA microarray 분석서비스 진행순서] 2주면 Profiling 완료!

더 높이 날고 싶지 않으십니까?

LNA™ 기술로 실험의 Quality를 높이세요

LNA™는 ribose ring을 methylen bridge로 연결

하여 구조를 고정시킨 핵산이다.

DNA, RNA oligonucleotide에 LNA를 삽입하면,

상보서열과의 결합력이 향상되어 최고의 특이성,

민감도를 보인다.

Locked Nucleic Acid (LNA™)란? LNA™ 적용분야 (Application)

Microarray analysis

Real-time PCR / Conventional PCR

SNP detection/allele specific PCR

Methylation analysis

In situ hybridization / Northern blotting

Inhibition of RNA function 외 다수…

[LNA™를 이용한 자유로운 Tm값 조절]

왼쪽. LNA™ 삽입 개수가 증가함에 따라 DNA oligonucleotide의 Tm값이 증가, 오른쪽. LNA™의 삽입은 Tm값을 유지하면서 길이가 짧은 oligonucleotide를 합성 가능

LNA™ 를 이용한 microRNA microarray는 비교도 안 될 정도의

높은 민감성, 높은 특이성으로 신뢰성 높은 데이터를 약속합니다.

GC함량이 다양한 synthetic microRNA를 각각 LNA™ probe, DNA probe로 제작한 microarray로 분석을 실시하였다. DNA probe를 이용할 경우(오른쪽)

에 비하여 LNA™ probe를 이용하였을 경우 GC 함량에 관계없이 다양한 microRNA를 검출할 수 있었다 (왼쪽)

7

Tm normalization – robust detection regardless of GC contentThe Tm and therefore the affinity of an oligonucleotide duplex can be controlled by varying the LNA™ content. This feature can be used to normalize the Tm across a population of short sequences with varying GC-content. For AT-rich oligonucleotides, which have low Tm, more LNA™ is incorporated into the LNA™ oligonucleotide to raise the Tm of the duplex. This enables the design of LNA™ oligonucleotides with a narrow Tm range. This is beneficial for microarray, PCR and other applications where sensitive and specific binding to many different targets must occur under the same conditions simultaneously. The power of Tm normalization is demonstrated by the comparison of DNA and LNA™ probes for detection of microRNA targets with varyingCG content (Figure 4).

Superior single nucleotide discriminationIntelligent placement of LNA™ monomers ensures excellent discrimination between closely related sequences. Differences as small as one nucleotide can be detected. The difference in Tm between a perfectly matched and a mismatched target is described as the delta Tm. Incorporation of LNA™ in oligonucleotides can increase the delta Tm between perfect match and mismatch binding by up to 8 °C. The increase in delta Tm enables better discrimination between closely related sequences such as members of microRNA families.

Broad applicabilityThe affinity-enhancing effects of LNA™ give LNA™ oligonucleotides strand invasion properties making LNA™ excellent for in vivo applications. Incorporation of LNA™ into oligonucleotides further increases resistance to endo- and exonucleases which leads to high in vitro and in vivo stability.

Since the physical properties (e.g. water solubility) of these sequences are very similar to those of RNA and DNA, conventional experimental protocols can easily be adjusted to their use.

LNA™ for microRNA researchThe small sizes and widely varying GC-content (5-95 %) of microRNAs make them challenging to analyze using traditional methods. The use of DNA or RNA based technologies for microRNA analysis can introduce high uncertainty and low robustness because the melting temperature (Tm ) of the oligonucleotide/microRNA duplex will vary greatly depending on the GC content of the sequences. This is especially problematic in applications such as microarray profiling and high throughput experiments where many microRNA targets are analyzed under the same experimental conditions.These challenges in microRNA analysis can be overcome by using LNA™-enhanced oligonucleotides. By simply varying the LNA™ content, oligonucleotides with specific Tm can be designed, regardless of the GC-content of the microRNA. Exiqon has used the LNA™ technology to Tm-normalize primers, probes and inhibitors to ensure that they all perform well under the same experimental conditions (Figure 5). Another challenge of studying microRNAs is the high degree of similarity between the sequences. Some microRNA family members differ by a single nucleotide. LNA™ can be used to enhance the discriminatory power of primers and probes to allow excellent discrimination of closely related microRNA sequences.

miRNA GC content

miRNA GC content

LNA™ capture probe

GC

content [%]

80

70

60

50

40

30

20

Sign

al s

tren

gth

[log2

]

4,000

8,000

16,000

miR

-190

miR

-340

miR

-29C

miR

-410

miR

-148

b

miR

-302

c

miR

-10b

miR

-299

-5p

miR

-147

miR

-199

a-5p

miR

-151

-5p

miR

-551

a

miR

-604

miR

-593

*

miR

323-

5p

miR

-572

DNA capture probe

GC

content [%]

80

70

60

50

40

30

20

Sign

al s

tren

gth

[log2

]

4,000

8,000

16,000

miR

-190

miR

-340

miR

-29C

miR

-410

miR

-148

b

miR

-302

c

miR

-10b

miR

-299

-5p

miR

-147

miR

-199

a-5p

miR

-151

-5p

miR

-551

a

miR

-604

miR

-593

*

miR

323-

5p

miR

-572

Figure 4. The power of Tm normalization. The signal from DNA-based capture probes varies with GC content and results in poor detection of many microRNAs, whereas LNA™ probes offer robust detection of all microRNAs. Signal intensity from microarray experiments using LNA™-enhanced (blue) or DNA based (gray) capture probes. MicroRNA targets with varying GC content were added at 100amol each.

See how LNA™ works...Watch the LNA™ movie atwww.exiqon.com/e-talk

1. What is LN

A™

?

LNA™ offers significant improvement in sensitivity and specificity and ensures optimal performance for all microRNA targets.

7

Tm normalization – robust detection regardless of GC contentThe Tm and therefore the affinity of an oligonucleotide duplex can be controlled by varying the LNA™ content. This feature can be used to normalize the Tm across a population of short sequences with varying GC-content. For AT-rich oligonucleotides, which have low Tm, more LNA™ is incorporated into the LNA™ oligonucleotide to raise the Tm of the duplex. This enables the design of LNA™ oligonucleotides with a narrow Tm range. This is beneficial for microarray, PCR and other applications where sensitive and specific binding to many different targets must occur under the same conditions simultaneously. The power of Tm normalization is demonstrated by the comparison of DNA and LNA™ probes for detection of microRNA targets with varyingCG content (Figure 4).

Superior single nucleotide discriminationIntelligent placement of LNA™ monomers ensures excellent discrimination between closely related sequences. Differences as small as one nucleotide can be detected. The difference in Tm between a perfectly matched and a mismatched target is described as the delta Tm. Incorporation of LNA™ in oligonucleotides can increase the delta Tm between perfect match and mismatch binding by up to 8 °C. The increase in delta Tm enables better discrimination between closely related sequences such as members of microRNA families.

Broad applicabilityThe affinity-enhancing effects of LNA™ give LNA™ oligonucleotides strand invasion properties making LNA™ excellent for in vivo applications. Incorporation of LNA™ into oligonucleotides further increases resistance to endo- and exonucleases which leads to high in vitro and in vivo stability.

Since the physical properties (e.g. water solubility) of these sequences are very similar to those of RNA and DNA, conventional experimental protocols can easily be adjusted to their use.

LNA™ for microRNA researchThe small sizes and widely varying GC-content (5-95 %) of microRNAs make them challenging to analyze using traditional methods. The use of DNA or RNA based technologies for microRNA analysis can introduce high uncertainty and low robustness because the melting temperature (Tm ) of the oligonucleotide/microRNA duplex will vary greatly depending on the GC content of the sequences. This is especially problematic in applications such as microarray profiling and high throughput experiments where many microRNA targets are analyzed under the same experimental conditions.These challenges in microRNA analysis can be overcome by using LNA™-enhanced oligonucleotides. By simply varying the LNA™ content, oligonucleotides with specific Tm can be designed, regardless of the GC-content of the microRNA. Exiqon has used the LNA™ technology to Tm-normalize primers, probes and inhibitors to ensure that they all perform well under the same experimental conditions (Figure 5). Another challenge of studying microRNAs is the high degree of similarity between the sequences. Some microRNA family members differ by a single nucleotide. LNA™ can be used to enhance the discriminatory power of primers and probes to allow excellent discrimination of closely related microRNA sequences.

miRNA GC content

miRNA GC content

LNA™ capture probe

GC

content [%]

80

70

60

50

40

30

20

Sign

al s

tren

gth

[log2

]

4,000

8,000

16,000

miR

-190

miR

-340

miR

-29C

miR

-410

miR

-148

b

miR

-302

c

miR

-10b

miR

-299

-5p

miR

-147

miR

-199

a-5p

miR

-151

-5p

miR

-551

a

miR

-604

miR

-593

*

miR

323-

5p

miR

-572

DNA capture probe

GC

content [%]

80

70

60

50

40

30

20

Sign

al s

tren

gth

[log2

]

4,000

8,000

16,000

miR

-190

miR

-340

miR

-29C

miR

-410

miR

-148

b

miR

-302

c

miR

-10b

miR

-299

-5p

miR

-147

miR

-199

a-5p

miR

-151

-5p

miR

-551

a

miR

-604

miR

-593

*

miR

323-

5p

miR

-572

Figure 4. The power of Tm normalization. The signal from DNA-based capture probes varies with GC content and results in poor detection of many microRNAs, whereas LNA™ probes offer robust detection of all microRNAs. Signal intensity from microarray experiments using LNA™-enhanced (blue) or DNA based (gray) capture probes. MicroRNA targets with varying GC content were added at 100amol each.

See how LNA™ works...Watch the LNA™ movie atwww.exiqon.com/e-talk

1. What is LN

A™

?

LNA™ offers significant improvement in sensitivity and specificity and ensures optimal performance for all microRNA targets.

모두 검출 가능! 이런.. 겨우 이 정도 검출

6

At a glance• Excellent sensitivity - significantly increased sensitivity

compared to DNA and RNA• Uniform detection - robust detection of all microRNAs,

regardless of GC-content• Increased specificity - detection of single nucleotide

mismatches• High stability - superior binding to small RNAs in vivo and

in vitro• Excellent flexibility - can be used for a wide range of samples

including biofluids and FFPE

What is LNA™?Locked Nucleic Acids (LNA™) are a class of high-affinity RNA analogs in which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding (Figure 2). As a result, oligonucleotides containing LNA™ exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. For each incorporated LNA™ monomer, the melting temperature (Tm ) of the duplex increases by 2-8 ºC (Figure 3). In addition, LNA™ oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high Tm. This is important when the oligonucleotide is used to detect short or highly similar targets.

Since LNA™ oligonucleotides typically consist of a mixture of LNA™ and DNA or RNA, it is possible to optimize the sensitivity and specificity by varying the LNA™ content of the oligonucleotide. Incorporation of LNA™ into oligonucleotides has been shown to improve sensitivity and specificity for hybridization-based technologies including PCR, microarray and in situ hybridization.

What is LNA™ ?

DNA: atcg LNA: ATCG

tcgatcgattagctacgtacgta Tm: 60°C23-mer

tcgatcgattAgctacgtacgta Tm: 64°C23-mer

tcgatcGattAgctaCgtaCgta Tm: 78°C

tcgatcgattagctacgtacgta Tm: 60°C23-mer

23-mer

---atcgattAgctAcgta---- Tm: 60°C16-mer

---------- aGCtacGT----- Tm: 61°C8-mer

Shorter length, similar TmSame length, higher Tm

+ LNA™

DNA

DNA/LNA™

DNA/LNA™

DNA

DNA/LNA™

DNA/LNA™

Figure 2. The structure of LNA™. The ribose ring is connected by a methylene bridge (orange) between the 2’-O and 4’-C atoms thus “locking” the ribose ring in the ideal conformation for Watson-Crick binding. When incorporated into a DNA or RNA oligonucleotide, LNA™ makes the pairing with the complementary strand more rapid and increases the stability of the resulting duplex.

Figure 3. Replace DNA with LNA™ for higher Tm. On the left, progressive substitution of DNA nucleotides with LNA™ increases the melting temperature of the oligonucleotide while maintaining the recognition sequence and specificity of the probe. On the right, LNA™ substitutions allow shortening of the probe while maintaining the same Tm.

An LNA™ oligonucleotide offers substantially increased affinity for its complementary strand, compared to traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity and specificity and makes LNA™ oligonucleotides ideal for the detection of small or highly similar DNA or RNA targets.

LNA™-enhanced oligonucleotides can be designed to have a similar affinity towards all types of sequences regardless of the GC-content.

1. W

hat i

s LN

A™

?

6

At a glance• Excellent sensitivity - significantly increased sensitivity

compared to DNA and RNA• Uniform detection - robust detection of all microRNAs,

regardless of GC-content• Increased specificity - detection of single nucleotide

mismatches• High stability - superior binding to small RNAs in vivo and

in vitro• Excellent flexibility - can be used for a wide range of samples

including biofluids and FFPE

What is LNA™?Locked Nucleic Acids (LNA™) are a class of high-affinity RNA analogs in which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding (Figure 2). As a result, oligonucleotides containing LNA™ exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. For each incorporated LNA™ monomer, the melting temperature (Tm ) of the duplex increases by 2-8 ºC (Figure 3). In addition, LNA™ oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high Tm. This is important when the oligonucleotide is used to detect short or highly similar targets.

Since LNA™ oligonucleotides typically consist of a mixture of LNA™ and DNA or RNA, it is possible to optimize the sensitivity and specificity by varying the LNA™ content of the oligonucleotide. Incorporation of LNA™ into oligonucleotides has been shown to improve sensitivity and specificity for hybridization-based technologies including PCR, microarray and in situ hybridization.

What is LNA™ ?

DNA: atcg LNA: ATCG

tcgatcgattagctacgtacgta Tm: 60°C23-mer

tcgatcgattAgctacgtacgta Tm: 64°C23-mer

tcgatcGattAgctaCgtaCgta Tm: 78°C

tcgatcgattagctacgtacgta Tm: 60°C23-mer

23-mer

---atcgattAgctAcgta---- Tm: 60°C16-mer

---------- aGCtacGT----- Tm: 61°C8-mer

Shorter length, similar TmSame length, higher Tm

+ LNA™

DNA

DNA/LNA™

DNA/LNA™

DNA

DNA/LNA™

DNA/LNA™

Figure 2. The structure of LNA™. The ribose ring is connected by a methylene bridge (orange) between the 2’-O and 4’-C atoms thus “locking” the ribose ring in the ideal conformation for Watson-Crick binding. When incorporated into a DNA or RNA oligonucleotide, LNA™ makes the pairing with the complementary strand more rapid and increases the stability of the resulting duplex.

Figure 3. Replace DNA with LNA™ for higher Tm. On the left, progressive substitution of DNA nucleotides with LNA™ increases the melting temperature of the oligonucleotide while maintaining the recognition sequence and specificity of the probe. On the right, LNA™ substitutions allow shortening of the probe while maintaining the same Tm.

An LNA™ oligonucleotide offers substantially increased affinity for its complementary strand, compared to traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity and specificity and makes LNA™ oligonucleotides ideal for the detection of small or highly similar DNA or RNA targets.

LNA™-enhanced oligonucleotides can be designed to have a similar affinity towards all types of sequences regardless of the GC-content.

1. W

hat i

s LN

A™

?

+ LNA™

[MicroRNA microarray 검출 비교 : LNA™ vs DNA]