Conventional Banding Techniques Research Articles

First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand

The first chromosome study of Trigonostigma somphongsi (Meinken,1958) from Thailand. Specimens were collected from Pak Phli District, Nakhornnayok Province, central Thailand. The mitotic chromosomes were directly prepared from kidney tissues of two males and two females. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results shown that diploid chromosome number of T. somphongsi was 2n=50, the fundamental numbers (NF) were 92 in both male and female. The types chromosomes consisted of 10 metacentric, 20 submetacentric, 12 acrocentric and 8 telocentric chromosomes. The result also exhibited that the interstitial nucleolar organizer regions (NORs) were clearly observed at the short arm of chromosome pair 2. The karyotype formula could be deduced as: 2n (diploid) 50=10m+20sm+12a+8t.

Karyomorphological delineation, and the NOR loci on the sex chromosome in three species of Chrysopeleinid (Chrysopeleinae: Colubridae) from Thailand

Abstract. Donbundit N, Bausriyod P, Tanomtong A, Srisamoot N, Sumontha M, Thongnetr W, Patawang I, Supiwong W, Ditcharoen S, Muanglen N, Kaewmad P. 2022. Karyomorphological delineation, and the NOR loci on the sex chromosome in three species of Chrysopeleinid (Chrysopeleinae: Colubridae) from Thailand. Biodiversitas 23: 3813-3819. The description of chromosomal characteristics of three snakes from Thailand namely, Ahaetulla fusca, A. prasina, and Chrysopelea ornata was by using conventional staining and Ag-NOR banding techniques. The results showed that the same diploid chromosome number (2n) = 36 (16 macrochromosomes + 20 microchromosomes) and the fundamental number (NF) was 54 while the karyotype formulae in these three species are as follows: A. fusca; 2n(36) = Lm6 + Sm2+ Ssm4 + Sa2+ ZZ/ZW + 20 microchromosomes, A. prasina; 2n(36) = Lm6+ Sm6+ Sa2+ ZZ/ZW + 20 microchromosomes and C. ornata; 2n(36) = Lm4+ Lsm2+ Sm2 + Ssm4+ Sa2+ ZZ/ZW + 20 microchromosomes. Besides, the ZZ/ZW sex chromosome was observed in all species, whereas C. ornata was discovered the heteromorphic sex chromosome for the first time. Nucleolar organizer regions (NORs) are located on telomeric region of one pair in all species, while located on microchromosomes in A. fusca and A. prasina, whereas C. ornata was detected on sex chromosomes. Therefore, the Z chromosome revealed the NOR on the telomeric region of the long arm, while NOR of the W chromosome was located on the telomeric region of the short arm.

CHROMOSOMAL POLYMORPHISM IN BULGARIAN PATIENTS WITH REPRODUCTIVE PROBLEMS – ONE GENETIC CENTRE EXPERIENCE

Chromosomal polymorphism is described as normal variants at chromosomal regions with no impact on the phenotype but a possible correlation to infertility and recurrent spontaneous abortions. The aim of this study was to evaluate the effect of the chromosomal polymorphisms involved in families with reproductive failures in the Bulgarian population. Material and methods: A total of 1733 patients with unexplained reproductive failures who visited the Laboratory of Medical Genetics – Varna, Bulgaria, (2004 - 2019) were investigated by conventional cytogenetic analysis GTG and CBG differential banding techniques and analyzed at the resolution 400-550 GTG bands. Results: Chromosomal polymorphisms were found in 173 infertile patients (9,98%). The sex distribution was 6,52% males and 3,46% females. The most frequent finding was inv(9)(qh) (23,7%). The other chromosomal variants, which were found, consisted: 9qh+/- variants (15,1%); polymorphisms on the short arms of the acrocentric chromosomes (21,4%); 16qh+ (12,7%) and 1qh+ (6,9%). Y chromosome polymorphism was found in 27,4% of the males with polymorphisms. Two rare cases of polymorphism involving the centromere regions - 19qcenh+ and 20pcenh+ were also found. Conclusion: There is growing evidence that polymorphisms may have a clinical impact on fertility and could take part in the etiology of RF. In this study, we found a significantly high percentage of polymorphisms (9,98%) among the tested patients, and they were more common among males. The statistical significance of increased incidence of chromosome variations found in our study emphasizes the need for routine evaluation of their role in families with RF in our country.

A comparative chromosome study on five Minnow fishes (Cyprinidae, Cypriniformes) in Thailand

The cytogenetic comparisons of five Minnow species from Thailand were presented here, i.e., Devario regina, D. laoensis, Rasbora paviana, R. aurotaenia and Esomus metalicus. The mitotic chromosomes were prepared directly from renal cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results revealed that all Minnow fishes studied possessed the same diploid chromosome number (2n) as 50 chromosomes. The fundamental numbers (NF) of D. laoensis, D. regina, R. paviana, R. aurotaenia and E. metalicus are 100, 100, 98, 98, and 98 respectively. Their karyotypes composing of metacentrics-submetacentrics-acrocentrics-telocentrics were as follows: 6-12-32-0 in D. regina, 6-10-34-0 in D. laoensis, 8-16-24-2 in R. paviana, 8-16-24-2 in R. aurotaenia and 8-10-30-2 in E. metalicus. The Ag-NOR banding technique provides the nucleolar organizer regions (NORs) at subtelomeric region of the short arm chromosome in the a submetacentric or acrocentric chromosomes that are located differently in the different chromosome pairs among species.

First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand

This research was the first report on karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand. The 10 male and 10 female specimens were collected from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Andaman Sea, Thailand. Mitotic chromosomes were directly prepared from gill and kidney tissues. The chromosomes were stained by conventional Giemsa staining and Ag-NOR banding techniques. Results showed that the diploid chromosomes number of A. gahhm was 2n=48, the fundamental numbers (NF) was 54 in both male and female. The karyotype consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. None of strange size chromosomes related to sex was found. The heteromorphic nucleolar organizer regions (NORs) were observed on telomeric short arm of first acrocentric which can defined as 1a1b. There is NOR in 1a and not in 1b. The karyotype formula of black surgeon fish was as follows: 2n (48) = La6+Lt20+Mt18+St4

Comparative karyotype study of three Cyprinids (Cyprinidae, Cyprininae) in Thailand by classical cytogenetic and FISH techniques.

Three species of ornamental fishes in the subfamily Cyprininae (family Cyprinidae) namely, Epalzeorhynchos frenatum (Fowler, 1934), Puntigrus partipentazona (Fowler, 1934), Scaphognathops bandanensis Boonyaratpalin et Srirungroj, 1971 were studied by classical cytogenetic and fluorescent in situ hybridization (FISH) techniques. Chromosomes were directly prepared from kidney tissues and stained by using conventional and Ag-NOR banding techniques. Microsatellite d(CA)15 and d(CGG)10 probes were hybridized to the chromosomes of three cyprinids. The results show that the three cyprinid species share the same diploid number as 2n=50 but there are differences in the fundamental number (NF) and karyotypes i.e. E. frenatum: NF = 78, 18m+10sm+10st+12a; P. partipentazona: NF = 80, 6m+24sm+14st+6a; S. bandanensis: NF = 66, 4m+12sm+34a. NOR positive masks were observed at the regions adjacent to the telomere of the short arm of the chromosome pairs 10 (submetacentric) and 1 (metacentric) in E. frenatum and P. partipentazona, respectively whereas those were revealed at telomeric regions of the long arm of the chromosome pair 9 (acrocentric) in S. bandanensis. The mapping of d(CA)15 and d(CGG)10 microsatellites shown that hybridization signals are abundantly distributed in telomeric regions of several pairs except d(CA)15 repeats in S. bandanensis, which are distributed throughout all chromosomes and d(CGG)10 repeats in P. partipentazona display the high accumulation only in the first chromosome pair.

Karyotype analysis and nucleolar organizer regions (NORs) of Sundaic yellow catfish, Hemibagrus capitulum (Siluriformes, Siluridae) in Thailand

This is the first report on karyo-morphological details and characteristic of nucleolar organizer regions (NORs) of Sundaic yellow catfish, Hemibagrus capitulum (Siluriformes, Siluridae), from Sirindhorn Peat Swamp Forest, Narathiwat Province, Thailand.The mitotic chromosome preparations were prepared from kidney cells of ten male and eight female specimens. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of H. capitulum was 2n = 56 and the fundamental number of both sexes was 108. The karyotype consists of 2 large metacentric, 8 large submetacentric, 2 large acrocentric, 18 medium metacentric, 12 medium submetacentric, 6 medium acrocentric, 4 medium telocentric, and 4 small metacentric chromosomes. None of strange size chromosomes related to sex was found. In addition, the telomeric NORs were clearly observed in the short arm of the submetacentric chromosome pair 16. The karyotype formula for H. capitulum is as follows: 2n = 56 = L2m + L8sm + L2a + M18m + M12sm + M6a + M4t + S4m.

5-Methylcytosine-Rich Heterochromatin in Reptiles

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

Additional description of karyotype and meiotic features of Takydromus sexlineatus (Squamata, Lacertidae) from northeastern Thailand

This study determined the standardized karyotype and idiogram of Takydromus sexlineatus from Surin Province in northeastern Thailand. Mitotic metaphase chromosomes from bone marrow cells and different meiotic stage from testicular cells respectively were studied after staining with conventional Giemsa and Ag-NOR banding techniques for chromosome identification. The study was carried out with samples taken from both male and female T. sexlineatus, the sex of which was identified by external examination. The results showed that the diploid chromosome number of T. sexlineatus was 2n = 38 and the fundamental number (NF) was 38 in both male and female. Although chromosome study was done in both sexes of T. sexlineatus, this study does not identify the sex chromosomes. This is because of the limitation of the staining techniques used. Therefore, we did not conclude on the sex chromosome system in this species and all chromosomes were taken as autosomes. The types of autosomes observed were 16 large telocentric, 14 medium telocentric, and 6 small telocentric macrochromosomes and 2 microchromosomes. Our result is the new record in Thailand on this species and NOR characteristics by silver staining is the first report on NOR banding in this species. The karyotype formula of T. sexlineatus thus was found as: 2n(38) = L 16 t + M 14 t + S 6 t + 2 microchromosomes.

A Report on Classical Cytogenetics of Hihgfin Barb Fish, <i>Cyclocheilichthys armatus</i> (Cypriniformes, Cyprinidae)

Conventional staining and Ag-NORs banding techniques were applied on the chromosomes of hihgfin barb fish, Cyclocheilichthys armatus. Specimens were collected from the Chao Phraya River, Thailand. The mitotic chromosome preparations were directly obtained from kidney tissues of eight males and eight females. The results revealed that the diploid chromosome numbers of C. armatus was 2n=50 and the fundamental number (NF) was 94 in both males and females. The karyotype consists of four large metacentric, six large submetacentric, two large acrocentric, eight medium metacentric, 12 medium submetacentric, 12 medium acrocentric and six medium telocentric chromosomes. No sex chromosome was observed. Ag-NORs indicating NORs were clearly exhibited at the region adjacent to telomere of the short arm of some chromosomes. The NORs located on chromosomes of the metacentric chromosome pair 3 and the submetacentric chromosome pair 7. The karyotype formula of C. armatus is 2n=50=Lm4+Lsm6+La2+Mm8+Msm12+Ma12+Mt6. Cytogenetic data of cyprinid fishes can be used as the basic knowledge for enhancing the existing cytotaxonomic information and chromosome evolution of the Cyprinidae especially in the genus Cyclocheilichthys.

Use of Array Comparative Genomic Hybridization for the Diagnosis of DiGeorge Syndrome in Saudi Arabian Population

DiGeorge syndrome (DGS) is a genetic disorder known as a clinically variable syndrome with over 180 associated phenotypic features. It is caused by a common human deletion in the 22q11.2 chromosomal region and currently is affecting approximately 1 in 4,000 individuals. Despite the prevalence of inherited diseases mainly due to consanguineous marriages, the current diagnosis of DGS in Saudi Arabia is mainly based on conventional high-resolution chromosome banding (karyotyping) and FISH techniques. However, advanced genome-wide studies for detecting microdeletions or duplications across the whole genome are needed. The aim of this study is to implement and use aCGH technology in clinical diagnosis of the 22q11.2 deletion in Saudi Arabian DGS patients and to confirm its effectiveness compared to conventional FISH and chromosome banding techniques. Thirty suspected DGS patients were assessed for chromosome 22q11.2 deletion using high-resolution G-banding, FISH, and aCGH. The aCGH results were compared with those obtained by the other 2 cytogenetic techniques. G-banding detected the 22q11.2 deletion in only 1 patient in the cohort. Moreover, it detected additional chromosomal aberrations in 3 other patients. Using FISH, allowed for detection of the 22q11.2 deletion in 2 out of 30 patients. Interestingly, the use of aCGH technique showed deletions in the chromosome 22q11.2 region in 8 patients, indicating a 4-fold increase in diagnostic detection capacity compared to FISH. Our results show the effectiveness of aCGH to overcome the limitations of FISH and G-banding in terms of diagnostic yield and allow whole genome screening and detection of a larger number of deletions and/or duplications in Saudi Arabian DGS patients. Except for balanced translocations and inversions, our data demonstrate the suitability of aCGH in the diagnostics of submicroscopic deletion syndromes such as DGS and most chromosomal aberrations or complex abnormalities scattered throughout the human genome. Our results recommend the implementation of aCGH in clinical genomic testing in Saudi Arabia to improve the diagnostic capabilities of health services while maintaining the use of conventional cytogenetic techniques for subsequent validation or for specific and known aberrations whenever required.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 21

Small supernumerary marker chromosomes (sSMCs) are small supernumerary aberrant chromosomes that are generally equal in size or smaller than a chromosome 20, and cannot be identified or characterized by conventional cytogenetic banding techniques [1–3]. sSMCs can appear in 0.044% of newborn infants and in 0.075% of prenatal cases [1,3,4]. About 70% of sSMCs are caused by a de novo event [5], about 70% of sSMCs are originated from acrocentric chromosomes [1,6], and about 70% of de novo sSMCs are associated with no phenotypic effects [4]. Prenatal diagnosis of sSMCs gives rise to difficulties in genetic counseling, and identification of the nature of the aberrant chromosome requires molecular cytogenetic technologies [4,7–10]. We present our experience of the prenatal diagnosis and molecular cytogenetic characterization of an sSMC derived from chromosome 21 using fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). A 36-year-old woman, gravida 2, para 1, underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed an sSMC, which was C-band positive and nucleolar organizing region-stain positive. The parental karyotypes were normal. The karyotype was 47,XX,+mar (Figure 1). The sSMC hybridized with a centromere 13/21-specific α-satellite DNA probe (D13Z1/D21Z1) (cep13/21) (Cytocell, Adderbury, Oxfordshire, UK) (Figure 2). MLPA was used to determine the origin of the sSMC using a SALSA MLPA P181 centromere kit (MRC-Holland, Amsterdam, the Netherlands) (Figure 3). The results of MLPA indicated a duplication of the 21q11.2 segment containing the STCH and SAMSN1 genes. FISH determination of the duplications of the STCH and SAMSN1 genes was performed using the bacterial artificial chromosome clone probes RP11-138O15 and RP11-392H8 at 21q11.2. FISH determination of 21q21.1 involvement utilized the 21q21.1-specific bacterial artificial chromosome clone probes RP11-89M24 and RP11-109H14. FISH revealed STCH and SAMSN1 gene duplications in the fetus with sSMC(21) (Figure 4). No RP11-89M24or PRENATAL DIAGNOSIS AND MOLECULAR CYTOGENETIC CHARACTERIZATION OF A SMALL SUPERNUMERARY MARKER CHROMOSOME DERIVED FROM CHROMOSOME 21

Karyotypic study of five Lutjanid species using conventional and Ag-NORs banding techniques

The first cytogenetic comparisons of five snapper species from Thailand were presented here. Renal cell samples were taken from blacktail snapper (Lutjanus fulvus), five lined snapper (L. quinquelineatus), dory snapper (L. fulviflamma), brownstripe red snapper (L. vitta), and mangrove red snapper (L. argentimaculatus). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results exhibited that all five snapper species have the diploid chromosome numbers of 2n = 48 and the fundamental numbers (NF) of 48. The presences of large, medium, and small telocentric chromosomes were 22-24-2, 24-20-4, 36-10-2, 28-16-4 and 36-10-2, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) at subcentromeric region of the long arm of the respective telocentric chromosome pairs 9, 1, 3, 4 and 9. Their karyotype formulas is as follows: L. fulvus (2n = 48): L 22 t + M 24 t + S 2 t , L. quinquelineatus (2n = 48): L 24 t + M 20 t + S 4 t , L. fulviflamma (2n = 48): Lt36 + Mt10 + St2, L. vitta (2n = 48): L 28 t + M 16 t + S 4 t , and L. argentimaculatus (2n = 48): L 36 t + M 10 t + S 2 t .

Cytogenetics of the skinks (Reptilia, Scincidae) from Thailand; III: the first karyological study of Sphenomorphus maculatus and Jarujinia bipedalis

AbstractThis is a karyological study of male and female Sphenomorphus maculatus from Khon Kaen Province in Northeastern Thailand, and female Jarujinia bipedalis from Kanchanaburi Province in Central Thailand. Chromosomes of two species were prepared by the squash technique from the bone marrow and testis. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosome with Giemsa’s solution and silver nitrate solution, respectively. The results showed that the diploid chromosome number of Sphenomorphus maculatus and Jarujinia bipedalis was 30 and the fundamental number (NF) was 44. There were eight large metacentric, two medium metacentric, four small metacentric and 16 microchromosomes in Sphenomorphus maculatus and eight large metacentric, six small metacentric, two small telocentric and 14 microchromosomes in Jarujinia bipedalis. There was no irregularly sized chromosome related to sex in either species. For Sphenomorphus maculatus, NORs are located at the telomere on the long ...

Karyotype and Idiogram of Indian Hog Deer (<i>Hyelaphus porcinus</i>) by Conventional Staining, GTG-, High-Resolution and Ag-NOR Banding Techniques

Karyotype and Idiogram of Indian Hog Deer (<i>Hyelaphus porcinus</i>) by Conventional Staining, GTG-, High-Resolution and Ag-NOR Banding Techniques

Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

In the present study, conventional staining and NORs banding as well as Fluorescence in situ hybridizat ion (FISH) using the 18S rDNA and telomeric (TTA GGG)n probes were applied to stain the chromosomes of crocodile catfish, Bagariussuchus (Siluriformes, Sisoridae) fro m the Chao Praya River, Thailand. Kidney cells of 6 male and 6 female crocodile catfishes were used as a sample. The mitotic chro mosome preparations were done directly fro m kidney cells. The results showed that the diploid chro mosome number of B. suchus was 2n=56, the fundamental numbers (NF) were 102 in both male and female. The karyotype comprises 17m+17sm+12a+10t. The nucleo lar organizer reg ions (NORs) were detected by Ag -NORs banding and 18S rDNA probe mapping. The 18S rDNA are terminally located on the short arm adjacent to the telomere of the single pair of the 1st chromosome pair whereas NOR-bearing chro mosome is only one chromosome of the 1st chromosome pair (1a 1b, poly morphic characteristic) at the subtelomeric region of the short arm. Moreover, FISH with telomeric probe showed hybridizat ion signals on each telomere of all chro mosomes and interstitial telomeric sites were not detected. There were variations in signals of FISH and their position in the karyotype along with variat ion in DNA sequences. These markers are useful for future discrimination of population of closely related species and their poly morphis m.

New record on karyological analysis and first study of NOR localization of parthenogenetic brahminy blind snake, Ramphotyphlops braminus (Squamata, Typhlopidae) in Thailand

This is chromosomal analysis of the brahminy blind snake, Ramphotyphlops braminus (Daudin, 1803), a newly recorded species in northeastern Thailand. Mitotic chromosomes were prepared directly from intestinal epithelia cells of five brahminy blind snakes. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the triploid chromosome number was 3n = 42, while the fundamental number (NF) was 78. The types of chromosomes were 24 macrochromosomes (19 m + 5sm) and 18 microchromosomes (9 m + 3sm + 6 t). The region adjacent to the short arm near the telomere of one in macrochromosome pair 8 showed a clearly observable nucleolar organizer region/NOR. There were no irregularly sized chromosomes related to sex. The karyotype formula for R. braminus is as follows: 3n (42) = Lm 12 + Sm 7 + Ssm 5 + 18 microchromosomes.

Precision in chromosome identification with leads in molecular cytogenetics: An illustrated review.

Chromosomal aberrations are a major cause of human genetic diseases. Conventional cytogenetic banding techniques are the method of identification for both numerical and structural chromosomal abnormalities but with limited resolution. However, precise identification and characterization of the chromosomal abnormalities can only be achieved by advanced molecular cytogenetic techniques. These techniques are based mainly on fluorescence in situ hybridization, which have become an invaluable tool in the field of diagnostics. The advent of these molecular cytogenetic techniques has helped in the identification of chromosomal abnormalities to its minutest level. Apparently, the leads in molecular cytogenetic techniques have paved way for advanced molecular diagnosis, which now plays a significant role in both diagnostics and clinical research. These advances have led to the increased knowledge of the possible molecular mechanism involved in the chromosomal rearrangements and the genotype-phenotype correlation thus helping the patients towards better diagnosis and genetic counseling. This article highlights the advances in molecular cytogenetic techniques emphasizing the precision in identification of chromosomal rearrangements, and also illustrates few chromosomal abnormalities pediatric cases identified using these molecular cytogenetic techniques.